Nikki D. Charlton

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.

Biodiversity of fungal endophyte communities inhabiting switchgrass (Panicum virgatum L.) growing in the native tallgrass prairie of northern Oklahoma

This study was conducted to explore fungal endophyte communities inhabiting native switchgrass plants from the tallgrass prairie of northern Oklahoma. The primary focus was to isolate these endophytes in pure culture from surface-sterilized plant tissues and provide taxonomic identifications based on comparative analysis of ITS rDNA gene sequences. From these data, we evaluated the biodiversity of these potentially beneficial endosymbionts from this rapidly disappearing habitat of the Great Plains. While important from a strictly conservationist standpoint, this survey further allowed us to identify candidate endophytes for introduction into commercial switchgrass cultivars for biomass enhancement. A total of 210 whole plant samples were collected at early vegetative, full reproductive and senescence stages. Fungal endophytes were isolated, identified to species level when possible, and grouped into communities based on plant part, collection month and part of the prairie from which the plants were collected. Species diversity for each community was estimated by Shannon diversity index, and differences in diversity indices were compared using a t-test. The presence of fungal species representing at least 18 taxonomic orders suggests a high level of diversity in switchgrass endophyte communities. The fungal communities from shoot tissue had significantly higher species diversity than communities from the root tissue. The abundance of taxa assigned to the order Hypocreales (to which mutualistic, clavicipitaceous endophytes of cool-season grasses belong) found in shoot (64%) and root tissues (39%) throughout the growing season suggests great potential for utilizing these endophytes for enhancing biomass production and stress resistance of this important bioenergy crop.

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.

The mycorrhizal fungus, Sebacina vermifera, enhances seed germination and biomass production in switchgrass (Panicum virgatum L).

Seed dormancy and slow seedling establishment are two major concerns in switchgrass (Panicum virgatum L.) production, often resulting in a poor stand with reduced productivity. Studies were conducted to investigate the stability of artificial associations between switchgrass and the ectomycorrhizal fungus, Sebacina vermifera, and to evaluate the potential benefits of this novel association in seed germination and biomass production. All six strains of S. vermifera tested had a high frequency of colonization on switchgrass roots of a synthetic cultivar NF/GA-993. The positive effects of the associations were reflected in plant height, root length, and biomass production. Inoculated plants produced as much as 75%, 113%, and 18% more shoot biomass than un-inoculated control plants in the first, second, and third harvest, respectively, with no consequent reduction in root biomass. Further, culture filtrates from some strains of S. vermifera increased seed germination in the switchgrass cultivar Kanlow by 52% over the control (p < 0.05). This study illustrates the great potential of microbial associations to increase biomass production and productivity of switchgrass.

Genetics, Genomics and Evolution of Ergot Alkaloid Diversity

The ergot alkaloid biosynthesis system has become an excellent model to study evolutionary diversification of specialized (secondary) metabolites. This is a very diverse class of alkaloids with various neurotropic activities, produced by fungi in several orders of the phylum Ascomycota, including plant pathogens and protective plant symbionts in the family Clavicipitaceae. Results of comparative genomics and phylogenomic analyses reveal multiple examples of three evolutionary processes that have generated ergot-alkaloid diversity: gene gains, gene losses, and gene sequence changes that have led to altered substrates or product specificities of the enzymes that they encode (neofunctionalization). The chromosome ends appear to be particularly effective engines for gene gains, losses and rearrangements, but not necessarily for neofunctionalization. Changes in gene expression could lead to accumulation of various pathway intermediates and affect levels of different ergot alkaloids. Genetic alterations associated with interspecific hybrids of Epichloë species suggest that such variation is also selectively favored. The huge structural diversity of ergot alkaloids probably represents adaptations to a wide variety of ecological situations by affecting the biological spectra and mechanisms of defense against herbivores, as evidenced by the diverse pharmacological effects of ergot alkaloids used in medicine.

Epichloë canadensis, a new interspecific epichloid hybrid symbiotic with Canada wildrye (Elymus canadensis)

Many Epichloë endophytes found in cool-season grasses are interspecific hybrids possessing much or all of the genomes of two or three progenitors. Here we characterize Epichloë canadensis sp. nov., a hybrid species inhabiting the grass species Elymus canadensis native to North America. Three distinct morphotypes were identified that were separated into two groups by molecular phylogenetic analysis. Sequence analysis of the translation elongation factor 1-α (tefA) and β-tubulin (tubB) genes revealed two copies in all isolates examined. Phylogenetic analyses indicated that allele 1 of each gene was derived from Epichloë amarillans and allele 2 from Epichloë elymi. This is the first documentation of an interspecific hybrid endophyte derived from parents of strictly North American origins. Alkaloid gene profiling using primers specific to genes in the peramine, loline, indole-diterpene and ergot alkaloid pathways may indicate chemotypic variation in the ergot alkaloid and loline pathways between the assigned morphotypes. All isolates have the gene enabling the production of peramine but lack genes in the indole-diterpene biosynthesis pathway. Morphology and phylogenetic evidence support the designation of isolates from El. canadensis as a new interspecific hybrid species.

Deletion of the Fungal Gene soft Disrupts Mutualistic Symbiosis between the Grass Endophyte Epichloë festucae and the Host Plant

ABSTRACT Hyphal anastomosis, or vegetative hyphal fusion, establishes the interconnection of individual hyphal strands into an integrated network of a fungal mycelium. In contrast to recent advances in the understanding of the molecular basis for hyphal anastomosis, knowledge of the physiological role of hyphal anastomosis in the natural habitats of filamentous fungi is still very limited. To investigate the role of hyphal anastomosis in fungal endophyte-plant interactions, we generated mutant strains lacking the Epichloë festucae soft (so) gene, an ortholog of the hyphal anastomosis gene so in the endophytic fungus E. festucae. The E. festucae Δso mutant strains grew similarly to the wild-type strain in culture but with reduced aerial hyphae and completely lacked hyphal anastomosis. The most striking phenotype of the E. festucae Δso mutant strain was that it failed to establish a mutualistic symbiosis with the tall fescue plant host (Lolium arundinaceum); instead, it killed the host plant within 2 months after the initial infection. Microscopic examination revealed that the death of the tall fescue plant host was associated with the distortion and disorganization of plant cells. This study suggests that hyphal anastomosis may have an important role in the establishment/maintenance of fungal endophyte-host plant mutualistic symbiosis.

Alkaloid Variation Among Epichloid Endophytes of Sleepygrass (Achnatherum robustum) and Consequences for Resistance to Insect Herbivores

Epichloid endophytes are well known symbionts of many cool-season grasses that may alleviate environmental stresses for their hosts. For example, endophytes produce alkaloid compounds that may be toxic to invertebrate or vertebrate herbivores. Achnatherum robustum, commonly called sleepygrass, was aptly named due to the presence of an endophyte that causes toxic effects to livestock and wildlife. Variation in alkaloid production observed in two A. robustum populations located near Weed and Cloudcroft in the Lincoln National Forest, New Mexico, suggests two different endophyte species are present in these populations. Genetic analyses of endophyte-infected samples revealed major differences in the endophyte alkaloid genetic profiles from the two populations, which were supported with chemical analyses. The endophyte present in the Weed population was shown to produce chanoclavine I, paspaline, and terpendoles, so thus resembles the previously described Epichloë funkii. The endophyte present in the Cloudcroft population produces chanoclavineI, ergonovine, lysergic acid amide, and paspaline, and is an undescribed endophyte species. We observed very low survival rates for aphids feeding on plants infected with the Cloudcroft endophyte, while aphid survival was better on endophyte infected plants in the Weed population. This observation led to the hypothesis that the alkaloid ergonovine is responsible for aphid mortality. Direct testing of aphid survival on oat leaves supplemented with ergonovine provided supporting evidence for this hypothesis. The results of this study suggest that alkaloids produced by the Cloudcroft endophyte, specifically ergonovine, have insecticidal properties.

Phylogenetic relatedness of the M2 double-stranded RNA in Rhizoctonia fungi

Isolates from closely related fungi in the Rhizoctonia species complex were examined for the occurrence of the M2 double-stranded RNA (dsRNA) by amplifying a conserved 1000 nucleotide region of the dsRNA with reverse transcription PCR. The M2 dsRNA was detected in representative isolates belonging to three anastomosis groups (AG) of R. solani (AG-1-IA, AG-4 and AG-6; teleomorph = Thanatephorus) and four AGs of binucleate Rhizoctonia (AGA, AG-F, AG-R and AG-U; teleomorph = Ceratobasidium). Amplified PCR products from the 3′ region of the M2 dsRNA from a representative sample of 12 isolates from eight different AGs were sequenced and subjected to parsimony analysis and coalescent simulations to infer ancestral lineages and to reconstruct the ancestral history of haplotypes. Seven dsRNA haplotypes were inferred from the sample of 12 isolates. One haplotype was composed of only isolates of Ceratobasidium belonging to different AGs. The rooted gene genealogies from coalescent simulations suggested that the ancestral M2 dsRNA haplotype most likely evolved in Thanatephorus (anamorph = R. solani AG-1-IA) and has been acquired recently by isolates of Ceratobasidium. Reconstruction of the ancestral history of haplotypes with a parsimony-based approach that assumes both mutation and recombination suggested that four haplotypes recombined before coalescing to their most recent common ancestor, while three haplotypes coalesced without recombination in the recent past. There was no unique association of haplotype within a specific AG of either Ceratobasidium or Thanatephorus to support co-evolution of the M2 dsRNA within the fungal host. To our knowledge this is the first report of a dsRNA occurring in Ceratobasidium that also is present in Thanatephorus.

Characterization of Epichloë coenophiala within the US: are all tall fescue endophytes created equal?

Tall fescue (Lolium arundinaceum) is a valuable and broadly adapted forage grass that occupies approximately 14 million hectares across the United States. A native to Europe, tall fescue was likely introduced into the US around the late 1800s. Much of the success of tall fescue can be attributed to Epichloë coenophiala (formerly Neotyphodium coenophialum) a seed borne symbiont that aids in host persistence. Epichloë species are capable of producing a range of alkaloids (ergot alkaloids, indole-diterpenes, lolines, and peramine) that provide protection to the plant host from herbivory. Unfortunately, most tall fescue within the US, commonly referred to as “Kentucky-31” (KY31), harbors the endophyte E. coenophiala that causes toxicity to grazing livestock due to the production of ergot alkaloids. Molecular analyses of tall fescue endophytes have identified four independent associations, representing tall fescue with E. coenophiala, Epichloë sp. FaTG-2, Epichloë sp. FaTG-3, or Epichloë sp. FaTG-4. Each of these Epichloë species can be further distinguished based on genetic variation that equates to differences in the alkaloid gene loci. Tall fescue samples were evaluated using markers to simple sequence repeats (SSRs) and alkaloid biosynthesis genes to determine endophyte strain variation present within continental US. Samples represented seed and tillers from the Suiter farm (Menifee County, KY), which is considered the originating site of KY31, as well as plant samples collected from 14 states, breeders seed and plant introduction lines (National Plant Germplasm System, NPGS). This study revealed two prominent E. coenophiala genotypes based on presence of alkaloid biosynthesis genes and SSR markers and provides insight into endophyte variation within continental US across historical and current tall fescue samples.