110. Clonal origin and phylogeny in Sclerotinia sclerotiorum.
Ignazio Carbone and Linda M. Kohn, Dept. of Botany, Univ. of Toronto, Erindale College, Mississauga, Ontario, Canada, L5L 1C6.
Sclerotinia sclerotiorum is a cosmopolitan ascomycetous plant pathogen with a wide host
range. This fungus reproduces asexually by sclerotia and sexually by self-fertilization. Both
mycelial compatibility testing and DNA fingerprinting with a dispersed nuclear repetitive element
have shown that S. sclerotiorum has a predominantly clonal population structure with many
unique genotypes. We are using a phylogenetic approach to study whether mutation or
recombination is the dominant evolutionary force that is giving rise to new genotypes. If mutation
is the diversifying force, then clones should be related to each other in clonal lineages and
phylogenies will be well-supported. If recombination is giving rise to new genotypes, then clonal
lineages should fall apart and phylogenies will be poorly resolved. So far we have identified three
phylogenetically informative regions in S. sclerotiorum, the intergenic spacer (IGS) of the nuclear
ribosomal DNA repeat, a portion of the gene encoding the translation elongation factor 1 alpha,
and one anonymous nuclear region. A parsimony analysis of nucleotide variation at all three loci
for a sample of 42 isolates of S. sclerotiorum from a Canadian canola field showed one most
parsimonious tree with a consistency index of 0.857. To further test our hypothesis of clonality,
we will compare the length of our tree with tree lengths obtained from randomized data sets and
also perform a likelihood analysis for each locus separately and for all three loci together. We are
also extending this study to other populations of S. sclerotiorum from both wild plants and other
agricultural hosts.
111. ITS sequences from the arbuscular mycorrhizal fungus, Gigaspora margarita.
Luisa Lanfranco, Silvia Perotto and Paola Bonfante. Dipartimento di Biologia Vegetale dell'Università and Centro Micologia del Terreno -C.N.R.- Viale Mattioli 25, 10125 Torino, Italy.
Arbuscular mycorrhizal fungi are obligate endosymbionts, colonizing the roots of almost
80% of land plants. They belong to the order Glomales, Zygomycetes, with at least 150 species.
Each asexual spore contains thousands of nuclei, with a relatively large amount of DNA (about
109 bp/nucleus) when compared to other fungi. Several PCR based-techniques have been recently
applied to explore the genetic diversity of these fungi. The ITS (Internal Transcribed Spacers) of
the nuclear ribosomal unit have proved to be very informative to reveal species-specific DNA
polymorphisms. It has been proved that a single spore of several Glomus species presents slighly
different ITS sequences. In order to study genetic variability and to devise molecular tools to
study species diversity in the field, we have investigated polymorphism of the ITS region of
different isolates of Gigasporaceae. Multispore DNA preparations were amplified with ITS1/ITS4
universal primers. Single bands were obtained, about 550-600 bp in size. The fragment of
Gigaspora margarita was cloned and after sequencing of different clones, at least three different
sequences were obtained. By comparison with sequences in data banks, two primers were
designed and tested for their capability to detect specifically G. margarita during the symbiotic
phase. A similar approach has been used to investigate the species Glomus versiforme.
112. Molecular phylogeny of the Agaricales based on 25S rDNA sequences.
Moncalvo, J. M., F. Lutzoni, S. Rehner, J. Johnson, and R. Vilgalys, Department of Botany, Duke University.
The identity of natural groups within the order Agaricales were addressed using ribosomal
DNA sequences. Approximately 900 bases of the 5' end of the nuclear-encoded large subunit
RNA gene (25S rDNA) were sequenced for over 300 taxa, selected broadly across all major
families within the Agaricales. A phylogenetic tree was estimated from 154 of the most diverse
taxa using maximum parsimony. Many groups were supported by moderate to high bootstrap
levels, or else are consistent with morphologically-based classification schemes. Some well
recognized groups were the families Amanitaceae, Coprinaceae (excluding C. comatus and
subfamily Panaelideae), and Agaricaceae (excluding the Cystodermateae), and the genera
Tricholoma, Termitomyces and its ally Podabrella, Pleurotus and Hohenbuehelia, etc.
Nonmonophyletic groups revealed were the families Tricholomataceae and Hygrophoraceae, and
the genera Clitocybe and Marasmius. This first-order estimate of phylogenetic relationships
among major lineages will serve as a starting point for further comparative studies on mushroom
biology and evolution.
113. Hypermutability as a source of genomic variation in the homothallic fungus Basidiobolus.
Rex Nelson and Bruce Cochrane. Department of Biology, University of South Florida, Tampa, FL 33620.
Members of the genus Basidiobolus (Ediam, 1886) are filamentous saprophytic fungi.
This genus has a sexual phase but is considered homothallic and as such, its reproduction is
essentially clonal. Genetic studies of isolates recovered from the environment demonstrate that
there exists a great amount of variation within and between populations of this fungus. RAPD
analysis of isolates recovered from the University of South Florida's Ecological Study Area
indicate that virtually each isolate is genetically unique. Sequencing of a fragment of the chitin
synthase gene revealed an extraordinarily high number of silent site differences among isolates. In
order to determine whether sequence variation is generated during sexual reproduction sequences
were obtained from single zygospore cultures derived from a single saprobic isolate. The
sequence obtained from one of the progeny isolates differs by 10% from the parental sequence.
All differences were silent with respect to amino acid sequence. Sequencing of a fragment of an
unrelated gene similar to the RNA binding domain of the RNP1 gene of Saccharomyces
cerevisiae reveal no sequence differeces between the parent and progeny. The spontaneous
mutation rate to resistance to miconazole was also determined from assay of conidia germination
on selectective media. This rate approached 2 x 10-5. Implications of these observations on the
genetic structure of Basidiobolus will be discussed.
114. Fungal pathovars from north American host species are distinct from those isolated from hosts not native to this continent.
Michael H. Perlin. University of Louisville, Louisville, KY 40292.
Molecular markers were used to compare haploid strains of Microbotryum violaceum
(Ustilago violacea) derived from different host species. This fungus is an heterobasidiomycete
which forms its spores in the flowers of over 200 species of Caryophylaceae (Pinks). On the
other hand, each strain of the fungus has a host range limited to one or a few plant species.
Although different M. violaceum strains, even from different hosts, are morphologically similar
and can apparently initiate mating, inter-pathovar matings seldom result in "normal" progeny;
often passage through host plants, when successful, leads to aneuploid or polyploid sporidia . In
order to establish relationships between and among different pathovars, haplotype analysis was
carried out on sporidia derived from eight different host species. Comparisons were made using
electrophoretic karyotypes and by phylogenetic analyses of two introns and an intervening exon of
their respective -tubulin genes. Strains from the same or similar host species had similar
chromosome profiles and similar, if not identical, DNA sequences in the region examined. Most
striking, were differences for both electrophoretic karyotype and DNA sequence polymorphisms
between the strains derived from North American host species and those from hosts native to
Europe. Thus, reproductive isolation has apparently resulted in major polymorphisms between
the North American M. violaceum strains and their European cousins.
115. Fungal Class C Cellulases.
Alan Radford, Biology Department, University of Leeds and Maristella Azevedo, Cell Biology Department, University of Brasilia.
We have now sequenced two CBH1 isozymes from Humicola grisea and one from Neurospora crassa and have a second equivalent N. crassa clone. Seventeen more fungal examples are sequenced, including both EG1 and CBH1 from Trichoderma reesei and other Trichoderma species, an EG1 from Humicola grisea, EG1 and CBH1 from Fusarium oxysporium, CBH1 from Penicillium janthinellum, Cochliobolus carbonum and Cryphonectria parasitica, and a series of CBH1 isozymes from Phanerochaete chrysosporium. Sequence alignment and phylogeny using CLUSTAL suggests that the ancestral class C cellulase was a CBH1 (exocellulase or cellobiohydrolase) which is found in both ascomycetes and basidiomycetes. By interstitial deletion, an EG1 (endocellulase) clade has arisen in ascomycetes and their now imperfect relatives, in T. reesei, T. longibrachiatum, H. grisea and F. oxysporium. The seven CBH1 isozymes of the basidiomycete P. chrysosporium form a single clade, and appear to have amplified within this line. The two CBH1 isozymes of H. grisea are closely related to each other, and to the single known CBH1 sequences from N. crassa and F. oxysporium. The single CBH1 of the two Trichoderma species, C. parasitica, C. carbonum and P. janthinellum forms a distinct branch. Most class C cellulases have a hinge region and a C-terminal cellulose-binding domain (CBD). However, these have been lost independently within several clades.