Inoculum 63(3) - Mycological Society of America

asymptomatic seedlings from seven plant species sampled at the Duke Forest
(NC) on multiple years. Surface sterilized plant tissue was used for fungal pure
culture isolation and total DNA extraction for community analysis. Taxonomic
identification of pure cultures was performed based on sequence information of
the internal transcribed spacer region (ITS). A collection of over 1300 pure cultures,
classified into 316 fungal taxa (97% similarity at the ITS region), was generated
from 320 seedlings. The most abundant fungal groups present in symptomatic
seedlings differed from those most commonly isolated from asymptomatic
seedlings and often represent known plant pathogenic groups. In addition, the
most abundant pathogen taxa were isolated from five of the seven host species
studied, and were isolated at lower frequency from asymptomatic seedlings. The
most abundant isolates from asymptomatic seedlings most closely matched samples
from GenBank with no clear taxonomic identification. For individual
seedlings studied, the fungal community appears to be simple at this stage, dominated
by one or two taxa. At the isolate level, no host-specificity was observed;
however, preliminary results from culture-independent analyses reveal community
structure separation according to host species. The implications of host specificity
of fungal pathogens in forest seedlings are discussed.
Berbee, Mary L 1 , Satoshi Sekimoto 1 , Ludovic LeRenard 1 , Joseph W Spatafora
2 , and AFTOL2 Working Group 1 . 1 Dept. of Botany, University of British Columbia,
Vancouver BC Canada, 2 Dept. of Botany and Plant Pathology, Oregon
State University, Corvallis OR USA. Becoming A Fungus: Comparative Phylogenetic
Studies of Evolution of Absorptive Nutrition
Most fungi gain nutrients by secreting digestive enzymes from their hyphae
into the surrounding matrix, and then absorbing the nutrients that diffuse
back. We are exploring data from our community sequencing proposal to the US
Joint Genome Institute to better understand the metabolic capabilities of early
fungi. With new genome sequences from early-diverging fungi including aquatic
chytrids and terrestrial zygomycetes, it becomes possible to compare secreted enzymes
across phyla and to speculate on the evolutionary origins of their underlying
genes. The enzymes that fungi secrete to break down cellulose or proteins
have paralogs that are not secreted but function instead in housekeeping within the
cell, in modifying polysaccharides or in recycling proteins. Duplication of the
housekeeping genes, modification for secretion, and addition of domains for binding
to particular substrates can result in enzymes adapted for extracellular function.
The Ascomycota and Basidiomycota share secreted enzymes with functions
including breaking down cellulose. The chytrids and zygomycetes also have the
housekeeping homologs to the secreted enzymes. However, true orthologs to the
secreted enzymes of the Ascomycota and Basidiomycota seem to be rare among
the early diverging clades. Assuming that it was the accumulation of plant biomass
on land that selected for the maintenance of the enzymes of the Ascomycota
and Basidiomycota, the absence of conserved secreted enzymes across other
phyla could be explained if other fungi diverged too early to encounter land plants
as a readily available source of nutrients.
Birkebak, Joshua M and Brandon Matheny. Department of Ecology and Evolutionary
Biology, University of Tennessee, 332 Hesler Biology Building
Knoxville, TN 37996-1610. A systematic revision of Clavariaceae (Agaricales)
from the Pacific Northwest
The diversity of Clavariaceae has been underestimated in the Pacific
Northwest of North America and thorough taxonomic revisions are much needed.
Molecular methods of species recognition have not been extensively used in
the Clavariaceae and are found to elucidate species level diversity previously
unidentified. Several species complexes exhibiting high morphological and sequence
diversity have been observed. Morphological differentiation of some molecularly
distinctive species is not yet possible, and cryptic species may be present.
A monographic revision of the Clavariaceae in the Pacific Northwestern North
America with taxonomic keys, illustrations and descriptions is being compiled
and will be published pending further investigation. At present, twenty-eight
species in the family have been identified based on herbarium and recent field collections.
Ten of these are tentatively considered undescribed. Overall, the Pacific
Northwest is represented by two species of Camarophyllopsis (one new), eight
species of Clavaria (one new), one species of Clavicorona, four species of
Clavulinopsis (two new), four species of Mucronella, and nine species of Ramariopsis
(six new).
Bittleston, Leonora S and Anne Pringle. Harvard University,16 Divinity Avenue
Cambridge, MA 02138. The insect and yeast communities of carnivorous
pitcher plants
Carnivorous pitcher plants are models for food web dynamics. The Northern
pitcher plant, Sarracenia purpurea, has modified leaves, or pitchers, which
are sterile until they open. Soon after opening the pitchers are filled with rainwater,
and accumulate a diverse community of organisms. The microbes in pitchers
are still relatively unknown, although recent studies have shown that a keystone
predator, the mosquito Wyeomyia smithii, controls bacterial diversity. Numerous
10 Inoculum 63(3), June 2012
yeasts have been found within the pitchers, and they are different from those present
in the surrounding bog water. We examined how insects affected the diversity
and abundance of yeasts in S. purpurea pitcher plants. Insect exclusion with
gauze coverings successfully excluded W. smithii, and the abundance of yeasts
was positively correlated with insect counts. The gauze treatment did not reduce
the abundance or diversity of yeasts in the pitchers, suggesting that insects introduce,
attract, or promote the growth of yeasts. Additionally, we found that one
commonly associated yeast, Candida globosa, is present internally in surfacesterilized
adult W. smithii, indicating that the pitcher plant mosquito acts as a vector
and may have a more complex association with this yeast. Convergently
evolved pitcher plants in the genus Nepenthes exist in Southeast Asia, and our preliminary
research show that there is also convergent community assembly of insect
and arachnid associates between Nepenthes and Sarracenia. These results
may extend to the communities of yeasts found within similar pitcher habitats on
opposite sides of the planet.
Blair, Jaime E and Nahill H Matari. Department of Biology, Franklin & Marshall
College, Lancaster, PA 17603. A timescale for Oomycete evolution estimated
from conserved regulators of gene expression
The fungal-like oomycetes are ubiquitous in nature, occupying niches in
marine, freshwater, and terrestrial ecosystems. While the diversity of saprophytic
oomycetes is most certainly underestimated, this group is primarily known for the
important plant and animal pathogens it contains. The oldest accepted fossil evidence
of biotrophic oomycetes associated with vascular plants comes from the
Early Devonian Rhynie chert (approx 400 Ma); however, the affinity of fossil
oomycetes to modern lineages remains unclear. Currently, genomic resources
exist for a number of oomycete species, as well as close relatives within the Stramenopiles
(diatoms, brown algae). The goal of this project was to use a comparative
genomics approach to identify conserved regulators of gene expression
within each of these genomes, and use Bayesian techniques to co-estimate phylogeny
and divergence times. We focused primarily on genes involved in key
processes such as RNA silencing and chromatin modification, as well as core eukaryotic
transcription factors. Oomycete genomes do contain the necessary complement
of genes for RNA silencing, including orthologs of Dicer, Argonaut,
dsRNA-binding proteins, and an RNA-dependent RNA polymerase; other researchers
have shown that these genes are expressed during various life stages in
Phytophthora infestans. Oomycetes also possess a full array of genes for histone
modification, including deacetylases, acetyltransferases, methyltransferases, and
chromodomain-containing proteins. Surprisingly, no homologs to canonical eukaryotic
DNA methyltransferases could be identified in the genomes of
oomycetes or in the outgroups. Bayesian divergence times were estimated from a
dataset of 53 proteins using conservative calibrations from the diatoms and the
Rhynie chert oomycetes. The sensitivity of estimated divergence times to model
specification on the prior parameters will also be discussed.
Blair, Jaime E, Lauren Coffua, Alison Greidinger, Amy Chabitnoy, and Lauren
Cook. Department of Biology, Franklin & Marshall College, Lancaster, PA
17603. Measuring oomycete biodiversity in aquatic, forest, and agricultural
ecosystems: culture-based and metagenomic approaches
The fungal-like Oomycota have long been studied by mycologists given
their osmotrophic lifestyle and habit of hyphal growth. Many species of
oomycetes are devastating pathogens, such as the notorious potato late blight
agent, Phytophthora infestans, and the cause of rare human pythiosis, Pythium insidiosum;
it is not surprising then that most research has focused on host-pathogen
interactions, with relatively little study concerning the natural diversity of saprotrophic
species. This knowledge gap has important ramifications on our estimates
of oomycete biodiversity, as well as on our understanding of the evolutionary history
of this important group. The goal of this study is to estimate and compare
oomycete biodiversity from several distinct habitats, including aquatic environments,
undisturbed forest soils, and highly managed agricultural settings. Our ongoing
culture-based surveys have relied on various baiting techniques and have
revealed a high level of diversity in aquatic and agricultural environments. However,
it has been shown that baiting methods can lead to biased estimates of biodiversity
as they tend to favor fast-growing organisms and those producing motile
zoospores. We have therefore developed a complementary metagenomic approach
using massively parallel pyrosequencing to more thoroughly sample
species diversity from the different environments. We are currently verifying two
markers for analysis, the mitochondrial cytochrome c oxidase subunit 1 (cox1)
locus and the nuclear ribosomal RNA internal transcribed spacers (ITS) and large
subunit (LSU) region; previous studies have shown that these loci are able to discriminate
among closely related species, and provide phylogenetic signal at a
number of taxonomic levels. We expect that this combination of culture-based
and sequence-based identification methods will enhance our understanding of
oomycete ecology and evolution, and perhaps give us more insight into the roles
of certain species in the outbreak of disease.
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