Kelly D. Craven

Prevalence of interspecific hybrids amongst asexual fungal endophytes of grasses

Epichloë endophytes are fungal symbionts of grasses that span a continuum including asexual mutualists that are vertically transmitted, obligately sexual pathogens that are horizontally transmitted, and mixed‐strategy symbionts with both mutualistic and pathogenic capabilities. Here we show that processes of genome evolution differ markedly for the different symbiont types. Genetic and phylogenetic analysis was conducted of a broad taxonomic, ecological and geographical sample of sexual and asexual isolates, in which were identified and sequenced alleles of genes for β‐tubulin (tub2) and translation elongation factor 1‐α (tef1), and microsatellite alleles were identified by length polymorphisms. The majority of asexual isolates had two or three alleles of most loci, but every sexual isolate had only single alleles for each locus. Phylogenetic analysis of tub2 and tef1 indicated that in all instances of multiple alleles in an isolate, the alleles were derived from different sexual species. It is concluded that, whereas horizontally transmissible species had haploid genomes and speciation occurred cladistically, most of the strictly seedborne mutualists were interspecific hybrids with heteroploid (aneuploid or polyploid) genomes. Furthermore, the phylogenetic evidence indicated that, in at least some instances, hybridization followed rather than caused evolution of the strictly seedborne habit. Therefore, the abundance of hybrid species among grass endophytes, and their prevalence in many host populations suggests a selective advantage of hybridization for the mutualistic endophytes.

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.

A Novel Test for Host-Symbiont Codivergence Indicates Ancient Origin of Fungal Endophytes in Grasses

Significant phylogenetic codivergence between plant or animal hosts (H) and their symbionts or parasites (P) indicates the importance of their interactions on evolutionary time scales. However, valid and realistic methods to test for codivergence are not fully developed. One of the systems where possible codivergence has been of interest involves the large subfamily of temperate grasses (Pooideae) and their endophytic fungi (epichloae). These widespread symbioses often help protect host plants from herbivory and stresses and affect species diversity and food web structures. Here we introduce the MRCALink (most-recent-common-ancestor link) method and use it to investigate the possibility of grass-epichloë codivergence. MRCALink applied to ultrametric H and P trees identifies all corresponding nodes for pairwise comparisons of MRCA ages. The result is compared to the space of random H and P tree pairs estimated by a Monte Carlo method. Compared to tree reconciliation, the method is less dependent on tree topologies (which often can be misleading), and it crucially improves on phylogeny-independent methods such as ParaFit or the Mantel test by eliminating an extreme (but previously unrecognized) distortion of node-pair sampling. Analysis of 26 grass species-epichloë species symbioses did not reject random association of H and P MRCA ages. However, when five obvious host jumps were removed, the analysis significantly rejected random association and supported grass-endophyte codivergence. Interestingly, early cladogenesis events in the Pooideae corresponded to early cladogenesis events in epichloae, suggesting concomitant origins of this grass subfamily and its remarkable group of symbionts. We also applied our method to the well-known gopher-louse data set.

New Neotyphodium endophyte species from the grass tribes Stipeae and Meliceae

Several species of Achnatherum (grass tribe Stipeae) and Melica (tribe Meliceae) typically are infected by nonpathogenic, seed-transmissible fungi with characteristics of Neotyphodium species (anamorphic Clavicipitaceae). Molecular phylogenetic studies clearly have distinguished the endophytes from Achnatherum inebrians (from Xinjiang Province, China), A. robustum and A. eminens (both from North America) and indicate that the A. inebrians endophyte comprises a unique nonhybrid lineage within the Epichloë and Neotyphodium phylogeny, whereas the endophytes of A. robustum, and A. eminens are hybrids with multiple Epichloë species (holomorphic Clavicipitaceae) as ancestors. Likewise distinct hybrid origins are indicated for Neotyphodium species from the European Melica species, M. ciliata and M. transsilvanica, the South African species M. decumbens and M. racemosa, and the South American species M. stuckertii. Neotyphodium species have been described from A. inebrians from Gansu Province, China, (N. gansuense), A. eminens (N. chisosum), M. stuckertii (N. tembladerae) and the South African Melica species (N. melicicola). However the endophytes from A. robustum and the European Melica species have not been described and the phylogenetic relationships of N. gansuense have not been investigated. Here we report a comprehensive study of morphological features and phylogenetic analyses of β-tubulin and actin gene sequences on an expanded collection of endophytes from the Stipeae and Meliceae. These data provide a firm foundation for the description of two new Neotyphodium species, N. guerinii from M. ciliata and M. transsilvanica, and N. funkii from A. robustum. We also propose the new variety, N. gansuense var. inebrians for endophytes of A. inebrians from Xinjiang Province, which are morphologically and phylogenetically distinct from, yet clearly related to, N. gansuense from Gansu Province.

At Death's Door: Alternaria Pathogenicity Mechanisms

Christopher B. Lawrence*, Thomas K. Mitchell, Kelly D. Craven, Yangrae Cho, Robert A. Cramer Jr. and Kwang-Hyung Kim Virginia Bioinformatics Institute and Department of Biological Sciences, Blacksburg, VA 24061, USA Department of Plant Pathology, Ohio State University, Columbus, OH 43210, USA Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA Department of Plant and Environmental Protection Sciences, University of Hawaii, Honolulu, HI 96822, USA Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717, USA (Received on April 14, 2008; Accepted on May 23, 2008)

Anastomosis is required for virulence of the fungal necrotroph Alternaria brassicicola.

ABSTRACT A fungal mycelium is typically composed of radially extending hyphal filaments interconnected by bridges created through anastomoses. These bridges facilitate the dissemination of nutrients, water, and signaling molecules throughout the colony. In this study, we used targeted gene deletion and nitrate utilization mutants of the cruciferous pathogen Alternaria brassicicola and two closely related species to investigate hyphal fusion (anastomosis) and its role in the ability of fungi to cause disease. All eight of the A. brassicicola isolates tested, as well as A. mimicula and A. japonica, were capable of self-fusion, with two isolates of A. brassicicola being capable of non-self-fusion. Disruption of the anastomosis gene homolog (Aso1) in A. brassicicola resulted in both the loss of self-anastomosis and pathogenicity on cabbage. This finding, combined with our discovery that a previously described nonpathogenic A. brassicicola mutant defective for a mitogen-activated protein kinase gene (amk1) also lacked the capacity for self-anastomosis, suggests that self-anastomosis is associated with pathogenicity in A. brassicicola.

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.

There are many ways to be a mutualist: Endophytic fungus reduces plant survival but increases population growth

One of the challenges to quantifying the costs and benefits of symbiosis is that symbionts can influence different components of host fitness. To improve understanding of the ecology of inherited symbionts, we developed general theory for a perennial host-hereditary symbiont interaction, in which symbionts can have independent and potentially opposing effects on host regeneration and survival. The model showed that negative effects on one component of fitness may be outweighed by positive effects on another, leading to a net positive impact of symbiosis on population growth. Model predictions depended on the availability of suitable patches, which influenced the relative contributions of survival vs. regeneration to host fitness. We then used experimental symbiont removal to quantify effects of a hereditary, fungal endophyte on a grass host. Endophyte presence strongly reduced host survival but increased regeneration. Application of the model revealed that negative effects on plant survival were overwhelmed by beneficial effects on regeneration, resulting in stable endophyte persistence at 100% frequency, consistent with field observations. Our work demonstrates the utility of a demographic perspective for predicting the dynamics of symbioses and supports the hypothesis that symbionts function as mutualists when host and symbiont fitness are coupled through vertical transmission.

The Small GTPase RacA Mediates Intracellular Reactive Oxygen Species Production, Polarized Growth, and Virulence in the Human Fungal Pathogen Aspergillus fumigatus

ABSTRACT Aspergillus fumigatus is the predominant mold pathogen in immunocompromised patients. In this study, we present the first characterization of the small GTPase RacA in A. fumigatus. To gain insight into the function of racA in the growth and pathogenesis of A. fumigatus, we constructed a strain that lacks a functional racA gene. The ΔracA strain showed significant morphological defects, including a reduced growth rate and abnormal conidiogenesis on glucose minimal medium. In the ΔracA strain, apical dominance in the leading hyphae is lost and, instead, multiple axes of polarity emerge. Intriguingly, superoxide production at the hyphal tips was reduced by 25% in the ΔracA strain. Treatment of wild-type hyphae with diphenylene iodonium, an inhibitor of NADPH oxidase, resulted in phenotypes similar to that of the ΔracA strain. These data suggest that ΔracA strain phenotypes may be due to a reduction or alteration in the production of reactive oxygen species. Most surprisingly, despite these developmental and growth abnormalities, the ΔracA strain retained at least wild-type virulence in both an insect model and two immunologically distinct murine models of invasive pulmonary aspergillosis. These results demonstrate that in vitro growth phenotypes do not always correlate with in vivo virulence and raise intriguing questions about the role of RacA in Aspergillus virulence.

Enhancement of switchgrass (Panicum virgatum L.) biomass production under drought conditions by the ectomycorrhizal fungus Sebacina vermifera.

ABSTRACT Experiments were conducted to examine the effects of cocultivating the important bioenergy crop switchgrass with the ectomycorrhizal fungus Sebacina vermifera under severe drought conditions. Plants cocultivated with the fungus produced significantly higher biomass and had a higher macronutrient content than uninoculated control plants under both adequately watered and drought conditions.