Population and Evolutionary Biology
88 Evolution and functional analysis of mating-type genes (MAT) in sexual (Cochliobolus) and asexual (Bipolaris) fungi. Saenz, G. S., Berbee*, M. L., and G. Turgeon. Department of Plant Pathology, Cornell University, Ithaca, NY 14853 *Department of Botany, University of British Columbia, Vancouver BC V6T 2C9
In heterothallic fungi, mating-type genes (MAT) determine whether a fungal individual can interact favorably with a compatible individual and thus begin the process that leads to sexual reproduction. Asexual fungi either lack the ability to undergo sexual reproduction, or sexual reproduction is cryptic. Since mating-type genes have been identified in asexual species, we can assess the mating potential of a presumably asexual fungus by direct examination of MAT. MAT genes of sexual Cochliobolus heterostrophus are well characterized and provide a basis for comparison with MAT genes from closely related asexual fungi, Bipolaris sacchari and B. sorghicola. We are taking a two-step approach towards determining whether Bipolaris spp. have the potential to undergo sexual reproduction: (1) We are sequencing both MAT1-1 & MAT1-2 and their flanking regions in these asexual fungi to detect accumulating mutations with respect to C. heterostrophus MAT. The accumulation of mutations may result in the loss of MAT gene function. (2) We will test function of the MAT genes from these asexual species by expressing them in a MAT deletion strain of sexual C. heterostrophus. If Bipolaris MAT genes are able to function in Cochliobolus, then this is evidence that Bipolaris MAT either maintains the ability to outcross or that the MAT genes are selectively maintained for other cellular functions.
89 Organization and polymorphism of mating-type genes from the bipolar mushroom Coprinus disseminatus. James, Timothy1, Kues, Ursula2, and Vilgalys, Rytas1. 1Department of Biology, Duke University, Durham, NC, USA 27708 2Institute of Microbiology, Swiss Federal Institute of Technology, CH-8092 Zurich, Switzerland
The inky-cap mushrooms in the genus Coprinus display an extreme diversity of mating systems ranging from completely non-outcrossing to elaborate genetic architectures designed to increase outbreeding efficiencies to nearly 100%, e.g. C. cinereus. While the majority of Coprinus species have heterothallic mating systems governed by two unlinked mating-type loci (i.e., tetrapolar mating systems), evolutionary reversions to a mating system controlled by a single locus have occurred (i.e., bipolar mating systems). In order to understand the genetic basis for changes between tetrapolar and bipolar mating determination in mushroom fungi, we are investigating the mating genes of the bipolar mushroom Coprinus disseminatus. Using a positional cloning method we have isolated C. disseminatus genes homologous to the A mating-type homeodomain-encoding genes of C. cinereus. Moreover, these genes segregate with mating-type as determined by pairing studies. A chromosome walk will be used to assess whether genes homologous to the C. cinereus B mating-type are physically linked to the A factor genes in C. disseminatus.
90 Phylogenetic relationships of a new species of Cylindrocladium that causes a blight disease on Buxus spp. with similar taxa, based on morphology and DNA sequences of internal transcribed spacers and beta-tubulin. Beatrice Henricot. The Royal Horticultural Society, Plant Pathology, Wisley, Surrey, UK
A severe blight disease of Buxus spp. was observed in the mid-90s in the UK and since 1998 has spread throughout the whole country. Cases in France, Italy, Belgium and Holland have also been reported. Diseased plants showed dark brown spots on the leaves, black streaks on the stems and severe defoliation. A species of Cylindrocladium was consistently isolated from diseased samples and inoculation assays confirmed it as the causal agent of the disease. The morphological description as well as the sequencing of the ITS spacers and the beta-tubulin gene showed that this fungus is a new Cylindrocladium species. This species was found to be the same as the one isolated from box plants in New Zealand and initially identified as Cylindrocladium spathulatum. The aim of this present study was to use these sequences to infer the phylogeny of this new species and other described Cylindrocladium species. An AFLP fingerprint technique was used to try to resolve genetic differences between isolates collected in different geographical locations in the UK and New Zealand. The origin of this new disease will be discussed.
91 Discordant gene genealogies and the evolution of the trichothecene gene cluster in Fusarium. Todd J. Ward1, H. Corby Kistler2, Joe Bielawski3, Eileen Sullivan1, and Kerry O'Donnell1. 1Microbial Properties Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois, USA 2Cereal Disease Laboratory, Agricultural Research Service, U.S. Department of Agriculture, St. Paul, Minnesota, USA 3University College, London, Department of Biology, London, England
During the last decade, fusarium head blight, or scab, reached epidemic proportions in the United States, resulting in over 2.6 billion dollars in losses to U.S. agriculture. Fusarium graminearum and closely related fungi are the causative agents of scab, and produce trichothecene mycotoxins that act as virulence factors on some hosts, and pose a serious threat to animal health and food safety. In order to investigate the evolution of trichothecene chemotypes within the genus Fusarium, and to provide an evolutionary framework for understanding the role of trichothecene mycotoxins in pathogenesis, a 19kb region of the trichothecene gene cluster (including 8 trichothecene genes) was sequenced in F. graminearum strains selected to represent the global genetic diversity of this pathogen. Comparisons of trichothecene gene-genealogies with each other and with those derived from genes outside the cluster, in conjunction with an examination of molecular evolutionary patterns, indicated that the species phylogeny is not representative of the evolutionary history of the trichothecene gene cluster. An evolutionary model incorporating multiple population level processes is discussed.
92 Multiple origins of serotype AD strains in the human pathogenic fungus Cryptococcus neoformans. Jianping Xu1, Rytas Vilgalys2, Guizhen Luo3, Mary Brandt4, and Thomas G. Mitchell3. 1Dept. of Biology, McMaster University, Canada; 2Dept. of Biology, 3Dept. of Microbiology, Duke University, USA, and 4Mycotic Diseases Division, CDC, Atlanta, USA
Cryptococcus neoformans is an important pathogenic basidiomycetous yeast of humans and other mammals throughout the world. Using commercial monoclonal antibodies to capsular epitopes, strains of C. neoformans manifest five distinct serotypes -- A, B, C, D, and AD. To understand the evolution of C. neoformans, and in particular, the origin(s) of serotype AD, we investigated 48 strains representing all five serotypes, including 17 different strains of serotype AD. For each of these strains, fragments of two genes (Laccase and Ura5) were sequenced and compared. All strains of serotypes A, B, C, and D, as well as three strains of serotype AD, had only one allele within each of the two loci. However, 14 of the serotype AD strains displayed two different alleles at each locus. Analysis of the sequences of each allele identified significant heterogeneity among the 17 serotype AD strains. Furthermore, significant sequence differences were observed between the two alleles within a locus in each of the14 serotype AD strains. Phylogenetic analysis revealed that one allele clustered with strains of serotype A and the other with strains of serotype D. The results suggest that serotype AD strains have multiple origins and support the hypothesis that 14 of the17 serotype AD strains originated from recent hybridizations between strains of serotype A and serotype D.
93 Mating-type gene organisation and field distribution in Discomycete Tapesia species. Paul S Dyer1, Greg Douhan2 and Tim D Murray2. 1School of Life and Environmental Sciences, University of Nottingham, Nottingham UK. 2 Washington State University, Pullman, WA USA.
Mating-type sequences have been cloned from the discomycete plant pathogen Tapesia yallundae. Two highly dissimilar DNA idiomorph regions, sizes 3.9 and 3.3 kb were detected in MAT-1 and MAT-2 isolates respectively, flanked by regions of nearly identical DNA sequence. Analysis of idiomorph sequences revealed the presence of two putative mating-type genes in the MAT- 1 idiomorph with conserved alpha-1 and high-mobility group (HMG) domains. An open reading frame for a putative metallothionein-like protein was also present, although this exhibited low homology. A single HMG domain mating-type gene was detected in the MAT-2 idiomorph. Knowledge of the mating-type sequences was used to devise a multiprimer PCR test for determination of mating-type of isolates of T. yallundae. This test employed three primers: a ææcommonÆÆ primer annealing to the idiomorph flanking region of both mating-types, and two ææspecificÆÆ primers annealing to sequences present in either the MAT-1 or MAT-2 idiomorphs. Locating the specific primers in different positions relative to the common primer yielded different size 811 bp or 417 bp products characteristic of MAT-1 or MAT-2 isolates respectively. The test was used to successfully determine the mating-type of 54 isolates of T. yallundae. It was also used successfully to determine the mating-type of isolates of the closely related eyespot pathogen T. acuformis. An investigation was made of the distribution of mating-types of T. acuformis at field sites worldwide in an attempt to determine possible reasons for the rare occurrence of the sexual cycle of T. acuformis compared to that of T. yallundae.
94 A multilocus molecular marker system for studying population subdivision in the rice blast fungus, Magnaporthe grisea. Brett C. Couch and Linda M. Kohn. Botany Department, University of Toronto, Mississauga, Ontario, Canada
The fungus, Magnaporthe grisea, is the causal agent of rice blast and gray leaf spot of grasses. It is one of the most important pathogens of rice due to its widespread occurrence and potential for serious crop losses when conditions are conducive to disease development. M. grisea comprises a number of host specific populations, based on studies utilizing DNA fingerprinting, RFLPs, and DNA sequence polymorphisms in the ITS region. As well, populations on rice are predominantly clonal based upon DNA fingerprinting studies. However, the possibility for sexual reproduction and recombination exists in populations on other grass hosts. These observations raise two interesting questions. First, how did rice -infecting populations originate? Second, are rice-infecting populations genetically isolated or are migration and gene flow occurring between rice-infecting populations and populations on other hosts? I have developed a molecular marker system suitable for addressing these questions. Fifteen polymorphic DNA genomic regions were identified by direct sequencing of these regions from a set of twenty-one reference isolates of M. grisea from rice and other grass hosts. Genomic regions were amplified for sequencing using previously published PCR primers or PCR primers designed from M. grisea sequences accessioned in Genbank and from the Clemson University M. grisea Genome Sequencing Project.
95 Identification of a novel invertase in the yellow ecotype of Neurospora intermedia. Alka Pandit and A. J. Griffiths. Department of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, B. C., Canada, V6T 1Z4.
The orange ecotype of N. intermedia is found on burnt plant material rich in sugar (like sugar cane), whereas, the yellow ecotype is commonly found on corn cobs. The absence of yellow stains from burnt substrates is striking. Both the ecotypes have similar requirements for growth under laboratory conditions, however, in nature they seem to have substrate preference. The reasons for this substrate preference are the focus of our investigation. Towards this goal morphological and cytological differences between the two ecotypes have been studied. The growth rates, conidial size and nuclear number per conidium have been compared. Both the ecotypes have similar growth rates but show differences in conidial size and nuclear number per conidium. The conidia of yellow ecotype are larger (approx. 1.6 times) than the orange ecotype and the number of nuclei in the conidia of yellow ecotype are about 3 times more than in the orange ecotype. The importance of these differences in adaptation to a particular substrate is unclear. As invertase may play a crucial role in colonisation and adaptation to a sugar rich substrate the invertase activity was assayed in both the ecotypes and the invertase isozyme was studied by gel electrophoresis. The specific activity of the intracellular invertase is more than 6 fold higher in the orange ecotype whereas the specific activity of the extracellular invertase is about 2 fold higher. Interestingly, the enzyme shows polymorphism that is consistent between the two ecotypes. The orange ecotype shows three bands for extracellular as well as the intracellular invertase. The invertase in yellow ecotype differ in the electrophoretic mobility and shows only two bands in extracellular invertase. The differences in the extracellular invertase tempt to suggest an adaptive role in nature. In order to characterise the invertase enzyme and the genes encoding it efforts are being made to amplify the invertase gene from both the ecotypes using PCR.
96 Relationship between genetic polymorphism and pathogenicity in Paracoccidioides brasiliensis. Flavia V. Morais1, Kátia C. Cândido 1, Patrícia S. Cisalpino2, Rosana Puccia1. 1Universidade Federal de São Paulo, São Paulo, Brasil. 2Universidade Federal de Minas Gerais, Belo Horizonte, Brasil.
Paracoccidioides brasiliensis is the temperature-dependent dimorphic fungus that causes paracoccidiodomycosis (PCM), a human systemic mycosis prevalent in Latin America. The major fungal antigen is the gp43, whose gene is located in a 1,329-bp DNA fragment including two exons, a 78-bp intron, and a leader peptide coding region of 105 bp. Here we describe polymorphism in the gp43 precursor gene after sequencing two PCR fragments from 17 P. brasiliensis isolates. The most polymorphic sequences showed 14 - 15 informative substitution sites compared with a consensus sequence and were phylogenetically distant from the others (with 1 - 4 informative sites). They encoded basic gp43 isoforms, generally neutral among the other isolates, and the three isolates in this group were from patients with pulmonary PCM. The biggest clade in a phylogenetic tree included the sequences of isolates from both lymphatic and pulmonary PCM. Preliminary data suggests that these samples are less pathogenic in mice infected i.p with 106 yeast forms, since the number of colony forming units in the spleen and liver was reduced when compared with that of mice infected with four other isolates. The gp43 promotor region (325 bp) showed little polymorphism among the 17 isolates analyzed (1 - 3 informative sites), and none was within transcription motifs. The P. brasiliensis isolates were grouped similarly according to the promotor and the gp43 precursor sequences. Sequencing data of the ribosomal ITS 1 and 2 regions of these isolates are being processed and will be compared. Supported by Fapesp, Pronex and CNPq
97 Phylogenetic characterization of two new isolates of Histoplasma capsulatum based on ITS and ETS region sequences from AIDS patients in Japan. Miki Tamura1, Takao Kasuga2, Kayo Watanabe1, Masakazu Katu1, Yuzuru Mikami1, and Kazuko Nishimura1. 1Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Chuo-ku, Chiba (260-8673), Japan 2 Roche Molecular Systems, 1145 Atlantic Ave, Alameda, CA 94501, USA
Histoplasma capsulatum is distributed worldwide and causes deep- mycosis in humans. In Asia, the number of cases of the disease histoplasmosis is increasing, however, very little is known about the population structure and pathogenicity of the pathogen. Recently we obtained two clinical isolates in Japan; one from Thai (IFM 49109), and other from Chinese (IFM 49110) AIDS patients. Random amplified polymorphic DNA (RAPD) analysis of both H. capsulatum strains showed that they have similar RAPD band patterns to the reference Asian strains, but their patterns were clearly different from those of a North American type 2 reference strain. In this research, we used DNA polymorphisms at the external transcribed spacer region (ETS) and internal transcribed spacer region (ITS) to study the population structure of H. capsulatum and to infer the origin of the two Japanese isolates. An unrooted dendrogram constructed from DNA sequences of internal transcribed spacer (ITS) of 27 geographically diverse H. capsulatum isolates representing two varieties, capsulatum and duboisii showed that the isolates could be classified into six clades; Asia type, South America types A and B, North America types 1 and 2, and H. duboisii type. The two Japanese isolates IFM 49109 and IFM 49110 were genetically close to North America type 1 group, but distinct. We judged that the two Japanese isolates were unique and thus created a new group the East Asia type. We also analyzed a part of the ETS region. ETS fragment was found to evolve faster and more informative than ITS regions. Dendrogram constructed from ETS sequences also showed that the two Japanese isolates were genetically unique among geographically diverse H. capsulatum isolates. DNA sequence analyses of ITS and ETS revealed the geographically differentiated population structure of H. capsulatum. Such information is essential to understand the epidemiology and evolution of the clinically important fungal pathogen.
98 Genetical and physiological diversity of Cladosporium spp. sympatrically colonising common reed (Phragmites australis). Stefan G.R. Wirsel1, Christiane Runge-Froboese 1, Dag G. Ahren2, Eric Kemen 1, Richard P. Oliver3 and Kurt W. Mendgen1. 1Lehrstuhl fuer Phytopathologie, Fachbereich fuer Biologie, Universitaet Konstanz, Universitaetstr. 10, D-78434 Konstanz, Germany 2Microbial Ecology, Ecology Building, Lund University, 22362 Lund, Sweden 3Australian Centre for Necrotrophic Fungal Pathogens, School of Biological Sciences, Murdoch University, Perth WA 6150, Australia
Species in the fungal genus Cladosporium exhibit diverse interactions with plants ranging from facultative biotrophy, to endo- and epiphyty. A collection of 44 isolates with characteristics of Cladosporium has been recovered from roots, stems and leaves of surface-sterilised common reed growing at Lake Constance (Germany). Morphological characterisation by high-resolution-cryo-SEM revealed that Cladosporium isolates from reed are diverse. Thereby, we distinguished three separate species, i.e. C. herbarum, C. oxysporium and Cladosporium sp. ITS sequence analysis supported these results and, moreover, separated the most common species, C. oxysporium, into two subclades. We have established two additional phylogenies in order to verify the placement of reed-associated Cladosporia and to improve the taxonomy of the genus as a whole. One differentiated fungi by their capacities to metabolise 95 different carbon sources using the BIOLOG microtiter system. The deduced phylogeny correlated with the morphological separations. The second phylogeny, based on actin-gene-sequences, showed the same four clades as did the ITS tree, but resulted in a higher resolution indicating putative cryptic species and a high diversity among Cladosporia at the population-species interface. Therefore, by using the phylogenetic species concept we propose that reed is colonised by at least four species of Cladosporium. A nested-PCR assay targeting variable sequences within actin introns indicated that these four sympatrically colonise reed. There was no evidence for mutual exclusion on or within the host or specialisation of these fungi for host habitats or organs. However, the incidence of colonisation increased during the season.
99 Stable polymorphism for the kalilo senescence plasmid in Hawaiian populations of Neurospora. Alfons Debets, Annelies van Mourik, Anthony J.F. Griffiths*, Rolf F. Hoekstra. Wageningen University, Genetics, Wageningen, The Netherlands. *UBC, Botany, Vancouver, BC, Canada
In this paper we describe the distribution of the kalilo senescence plasmid among 64 isolates of Neurospora intermedia and 64 isolates of N. tetrasperma from 42 soilsamples taken from Hawaii in 1998. We found that, though the frequency of a 'neutral' Hawaiian plasmid (Han- 2) was significantly lower, the kalilo frequency was similar to that found in 1972 and 1976 and there is still polymorphism for senescence in the N. intermedia population. The kalilo plasmid was also present in the N. tetrasperma population, and this was strongly correlated with senescence, demonstrating for the first time that kalilo-based senescence is not limited to the N. intermedia population of Hawaii. As compared to the N. intermedia population, kalilo isolates of N. tetrasperma are less frequent and have a longer lifespan. No spatial structuring was observed, there is co-occurrence of kalilo and non-kalilo isolates of both species.
100 Occurance of gray leaf spot disease of maize in east Africa. Okori, P., Fahleson. J, and Dixelius, C.. Dept. of Plant Biology, Swedish University of Agricultural Sciences, Box 7080, 750 07 Uppsala, Sweden.
Gray leaf spot disease of maize incited by Cercospora zeae-maydis (CZM) is today one of the biggest threats to maize production globally. In East Africa and other parts of the continent, the disease is now ranked second to maize streak gemini virus disease in economic importance. As such, studies of pathogen populations alongside resistance breeding are necessary. A hierarchical survey was used to collect CZM isolates in the major maize producing districts of Uganda, Kenya and Rwanda. Additionally, isolates from Zimbabwe and others previously used in phenetic studies in the United States were included in the study. Monoconidial cultures were then obtained from diseased leaf samples, transferred to potato dextrose broth media and grown for about two weeks. The mycelium was then freeze dried, DNA isolated and polymorphisms studied using AFLP (Amplified Fragment Length Polymorphism). The isolates clustered in two major groups, the African isolates clustered together with the few US group II isolates (NY2, OH8, VA1, GLS3 and GLS5) into one group while the US group I isolates (IN24, IL2, SCOH15 and GLS2) clustered into a small but distinct group. These results were in accordance with a previous investigation (Wang et al. 1998. Phytopathology 88:1269-1275) In general the isolates in their respective groups tended to exhibit growth and other cultural patters as earlier reported. This study support earlier obtained results and shows that group II isolates are prevalent in Eastern Africa while group I isolates was not found.
101 Neurospora in western North America: a model system in the backyard. David J. Jacobson1,2, Magdalen M. Barton2, Jeremy R. Dettman2, Megan D. Hiltz2, Amy J. Powell3, Gregory S. Saenz3, John W. Taylor2, N. Louise Glass2, and Donald O. Natvig3. 1Stanford University, Biological Sciences, Stanford, CA, USA. 2University of California, Plant and Microbial Biology, Berkeley, CA, USA. 3University of New Mexico, Biology, Albuquerque, NM, USA.
Species of Neurospora have been found mostly in the moist tropics and subtropics. Surprisingly, during the spring and summer of 2000, we observed Neurospora in the arid western United States as a primary colonizer of trees and shrubs killed by wildfires, significantly expanding the known geographic range and habitats of the genus. Neurospora colonies were observed in 23 forest fire sites in habitats ranging from cottonwood stands along the Rio Grande to mountain forests in New Mexico, the Sierra Nevada and Cascades in California, northeastern Nevada, Idaho, and northwest Montana to the Canadian border. Colonization occurred beneath the bark of diverse deciduous and conifer hosts. The combined 2000 collection includes 314 isolates from 35 to near 49 north latitude and from 750 m to >2400 m altitude. To date, 134 isolates have been identified to species; 130 (97%) are N. discreta. Within a site, mating type among individuals is often significantly skewed from a 1:1 ratio. The occurrence of Neurospora under these circumstances raises fundamental questions with respect to ecology and population biology: How does Neurospora gain access beneath apparently intact tree bark? How is it dispersed or vectored? How and where does it survive for decades between forest fires? What are the reproductive or genetic factors that cause the skewed mating type distribution? The 2000 collection provides a resource to begin addressing these questions.
102 The HET-s prion, a meiotic drive element causing sporekilling in Podospora anserina. H. Dalstra, F. Debets, K. Swart. Laboratory of Genetics, Wageningen University, The Netherlands.
P. anserina strains of het-s genotype exist as two phenotypes,the active [Het-s] and the neutral [Het-s*]. [Het-s] mycelium is vegetative incompatible with mycelium of the het-S genotype,whereas [Het-s*] is not.The [Het-s] character is transmitted as an infectious cytoplasmic element. [Het-s] is the prion form of the het-s encoded protein. In the classical model of sporekilling, the sporekiller is a segregation distorter, capable of aborting spores containing solely nuclei sensitive to killing. J. Bernet (1965) described a system similar to sporekilling. In a F[Het-s] X M[Het-S] cross at 18 C, up to 50% of the asci contained two normal and two aborted spores. In these asci, the two surviving spores were [Het-s] , whereas offspring from normal asci yielded two [Het-s*] and two [Het-S] spores. This system of sporekilling bears resemblance to the prion directed het-s/S vegetative incompatibility system. However in an encounter between [Het-s] and [Het-S] hyphae both sides are equal in hierarchy whereas during sexual crossing the difference in hierarchy is clear, the maternal mycelium being the only cytoplasm donor. In a F[Het-s]XM[Het-S] cross, the Het-S nuclei are possibly confronted with a prion permeated environment, potentially leading to an incompatibility reaction and subsequent abortion of young HET-S producing spores. The HET-s prion would act as a meiotic drive element in such a system. The work described in the poster focusses on the role the HET-s prion plays in the Het-sxHet-S type of sporekilling, as it emerges during sexual reproduction in P. anserina.
103 Quantitating the rate of concerted evolution in the ribosomal RNA multigene family. Austen R.D. Ganley1, 2Fred Dietrich and 1Rytas Vilgalys. 1Department of Biology and 2Department of Genetics, Duke University, Durham, NC27708
Concerted evolution describes the unusual evolutionary patterns of repetitive DNA elements whereby the repeats evolve together within a genome. These patterns arise through genomic mechanisms that maintain individual repeats with a consistent sequence, a process known as homogenization. Empirical determination of the homogenization mechanisms has remained elusive, primarily because an experimental system to test mechanisms is lacking. Similarly, the rate of homogenization is unknown and has been assumed to occur over evolutionary timescales. However recent work in fungi and other systems suggests that homogenization may occur relatively rapidly. We are investigating these issues using two approaches. In the first, Saccharyomyces cerevisiae is used as a model microevolutionary system to quantitate the rate of homogenization. We have introduced a small neutral change in the ribosomal RNA (rDNA) intergenic spacer, and are monitoring the spread of this "mutant" unit throughout the repeats in an array using a combination of quantitative PCR and pulsed field gels. Determination of a stable "normal" homogenization rate would allow us to use a genetic approach to test homogenization mechanisms. The second approach assesses the level of sequence diversity within an rDNA array using genomic sequencing data from several fungi (e.g. Ashbya, Cryptococcus, Candida). Sequence diversity is a product of the homogenization vs mutation rates, and so provides an indirect estimate of homogenization rate. However it may also be influenced by life cycle mode (e.g. sexual vs asexual reproduction). These experimental systems will aid interpretation of rDNA variability in fungi by providing insights into the dynamics of homogenization on a molecular level.
104 Analysis of genetic variation in Peronospora tabacinausing RFLPs. Serenella Sukno and Mark Farman. Dept. of Plant Pathology, University of Kentucky. S-305 Agricultural Sciences Center, North, Lexington, KY 40546-0091.
Peronospora tabacina Adam is the causal agent of blue mold of tobacco and belongs to the Oomycetes, a diverse group of fungus-like organisms that cause a wide range of destructive and economically important diseases on plants. The inability to identify and track specific P. tabacina populations hamper efforts to control blue mold. Such information is vital to the successful implementation of durable disease management strategies in US. To examine the genetics and population biology of this obligate biotrophic parasite, three PstI- genomic DNA libraries were constructed from DNA of three isolates that originated from Kentucky, USA. In preparation for a broader population study, 10 strains representing populations from Kentucky, Florida, Texas, Georgia, Pennsylvania, and Connecticut, were selected for an initial survey of RFLPs markers. PstI- and DraI- digested DNA were hybridized to 10 probes. Preliminary analysis indicates that there is a low level of genetic variation among the US populations. Two polymorphic probes were identified and seven different haplotypes could be differentiated among 10 isolates. Experiments are currently under way to evaluate the somatic stability of single spore lineages of one isolate and variability among individuals in a population. Results of the studies will be presented.
105 Ascospore morphology is a poor predictor of the phylogenetic relationships of Neurospora and Gelasinospora. Jeremy R. Dettman, Fred M. Harbinski, and John W. Taylor. Plant and Microbial Biology, University of California, Berkeley, CA, 94720
The genera Neurospora and Gelasinospora are morphologically comparable except the former produces ascospores with longitudinal elevated ridges (ribs) separated by depressed grooves (veins), and the latter genus produces ascospores with spherical or oval indentations (pits). Within a genus, the patterns of ascospore ornamentation can vary quite significantly between species, which suggests these unifying morphological characteristics may not be homologous. To assess the possibility of multiple independent origins of "ribbed" or "pitted" ascospores, the DNA sequences of four nuclear genes were obtained for 12 Neurospora taxa and four Gelasinospora taxa. Within the genus Neurospora, only three well-supported conclusions could be drawn: 1) the five outbreeding conidiating Neurospora species form a monophyletic group, 2) N. discreta is the most divergent of these five species, and 3) four homothallic Neurospora species form a monophyletic group. Evidently, the Neurospora and Gelasinospora taxa included in this study do not represent two clearly resolved monophyletic sister genera, but instead represent a polyphyletic group of taxa with close phylogenetic relationships and significant morphological similarities. The hypotheses of the monophyly of either pitted or ribbed ascospores could be rejected, which suggested that multiple origins of at least one of the character states were likely. Ascospore morphology, the character that the distinction between the genera Neurospora and Gelasinospora is based upon, was not an accurate predictor of phylogenetic relationships as inferred from the sequence data analyzed in this study.
106 Genetic variation of Colletotrichum lindemuthianum in bean fields. Raul Rodríguez-Guerra, Maria-Teresa Ramírez-Rueda, Octavio Martínez de la Vega and June Simpson. CINVESTAV, Unidad Irapuato, Apdo. Postal 629, Irapuato, Gto. Mexico.
Colletotrichum lindemuthianum is the causal agent of anthracnose in common bean (Phaseolus vulgaris). Analysis of monospore cultures from different bean fields in different regions of Mexico has shown that all such isolates can be distinguished at the genotype level using molecular markers but may often share pathotypes. The high level of variability is surprising since C. lindemuthianum is not known to undergo a sexual cycle under field conditions. Two commercial bean fields and one experimental plot were chosen in order to study variability in detail in terms of pathotype, capacity for anastomosis and molecular marker genotype of numerous isolates from each location. In addition one commercial field was sampled and analyzed in two consecutive years. Single spore isolates from a single plant were identical in terms of pathotype and capacity for anastomosis and the majority also had identical genotype patterns. A few isolates showed differences in a few bands. When single spore isolates from different individual plants were analyzed, all were found to have the same pathotype and very closely related or identical genotype patterns. In one commercial field and the experimental plot, differences could be determined in capacity for anastomosis where distinct groups could be determined in each case. In the commercial field where samples were taken in two consecutive years, in the second year although the capacity for anastomosis remained the same and genotpyes were also very similar, a new pathotypewas observed capable of infecting one extra cultivar of the differential set. We are grateful to CONACyT (K0195B) and SIHGO
107 Molecular clocks in Plectomycetes and the radiation of Histoplasma. Takao Kasuga1, Thomas J. White2 and John W. Taylor3. 1Roche Molecular Systems, Alameda, CA. 2Applied Biosystems, Foster City, CA. 3Plant & Microbial Biology, Univ. California, Berkeley, CA.
Owing to the scarcity of paleontological record, which is a prerequisite for calibration of time points, the nucleotide substitution rate in fungi has not been reported except for the small subunit ribosomal RNA gene (SSU rDNA). By using the published DNA substitution rate at the SSU rDNA as a time-standard together with pairwise DNA diversity data between closely related species, we estimated DNA substitution rates of seven independent protein-coding genes and the internal transcribed spacer (ITS) region in plectomycetes. Comparative analyses of multiple species showed that the DNA substitution rate in the class 1 chitin synthase gene was approximately constant across plectomycetous fungi, whereas that of ITS varied almost 10-fold. The estimated substitution rates at synonymous sites in protein coding genes ranged from 2.7x10-9 to 23.9x10-9 substitutions per site per year. These values are in the range of synonymous DNA substitution rates for a majority of protein coding genes in plants, animals and bacteria, despite of the enormous differences in body size, cellular organization, generation time and ecology.
A human pathogenic fungus Histoplasma capsulatum was believed to harbor three varieties, which showed differences in clinical manifestations and geographical distribution. DNA sequence variation in four independent protein genes revealed that H. capsulatum consisted of at least eight independent phylogenetic species. Combining the estimated DNA substitution rates with the phylogeny suggests that the radiation of Histoplasma started roughly 5 million years ago in South America.
108 Sexual recombination and dispersal of Cryptococcus neoformans var. gattii in the natural environment. Catriona Halliday and Dee Carter. University of Sydney, Microbiology, Sydney NSW Australia
Cryptococcus neoformans causes cryptococcosis in humans and animals, which is thought to begin by inhaling an infectious propagule from an environmental source. In Australia, C. neoformans var. gattii is most frequently isolated from Eucalyptus trees, which are thought to be the primary ecological niche of this variety. Understanding the occurrence of the fungus on host trees and its dispersal from these trees are likely to be important for assessing the risk of exposure to infectious propagules. We have therefore investigated the population structure of C. neoformans var. gattii isolates obtained from a number of host eucalypts within a limited geographical range. We began by assessing the potential for sexual recombination between isolates in this population, as dissemination is likely to occur via sexually produced basidiospores. The two mating types were found to be present in close to the 50:50 ratio expected by sexual outcrossing. However, when the structure of the population was assessed using AFLP loci a clonal pattern emerged. Finally, Canonical Analysis of Variance (CVA) of the AFLP dataset found a distinct division of genotypes according to their host tree, suggesting transmission between trees in this area is limited. We conclude that although the prevalence of C. neoformans var. gattii in this area is high and the potential for sexual recombination exists, the fungus does not commonly complete its lifecycle in association with the host trees but instead propagates as an asexual yeast that is not readily dispersed.
109 Cryptococcus neoformans: global molecular epidemiology. Wieland Meyer, Krystyna Maszewska, Mathew Hugnh and Sarah Kidd. Molecular Mycology Laboratory, Centre for Infectious Diseases and Microbiology, The University of Sydney at Westmead Hospital, Westmead, NSW, Australia
C. neoformans is a basidiomycetous yeast with three suggested varieties: var. grubii (serotype A), var. neoformans, (serotype D) and var. gattii (serotypes B and C). Varieties grubii/neoformans infect immunocompromised patients while variety gattii mainly infects immunocompetent hosts. The global genetic distribution of C. neoformans was studied by PCR-fingerprinting with single primers specific to minisatellite or microsatellite DNA. Clinical/environmental isolates obtained from around the world grouped into 8 major molecular types (VNI and VNII = serotype A, VNIII = serotype A/D, VNIV = serotype D and VGI, VGII, VGIII and VGIV = serotypes B and C). VNI and VGI were the most common genotypes. VGIII was geographically restricted to India/USA and VGIV to India/South Africa. Unique, strain-specific patterns were found for most of the US isolates, indicating a high degree of genetic diversity compared to isolates obtained from other areas in the world. Non-US isolates were highly genetically homogeneous or even clonal. When analysed with GelComparII the strains clustered broadly according to their country of isolation. Some strains were common to different countries. The overall global similarity between strains was 60%. Our findings support a division of C. neoformans into three varieties or even three separate species. Isolates obtained from the same patient at different time points and different body sites had identical banding patterns indicating a single source of infection. Regional profiles of eucalypt-derived and clinical isolates were concordant, supporting an epidemiological association between these trees and human infection. An automation of the methodology is currently underway.
110 Mitochondrial plasmids in Cryphonectria parasitica. Gobbi, E. and Rekab, D. University of Udine, DBADP, Udine, Italy.
The first plasmid reported in Cryphonectria parasitica was a mitochondrial (mt) plasmid pUG1, found in an Italian strain of the plant pathogen. It belongs to the small group of mt circular plasmids sharing no homology with the host DNAs together with plasmids of Neurospora spp., Pythium spp. and Absidia glauca. These plasmids propagate by using a plasmid encoded DNA polymerase whose features constitute and characterise a distinct subgroup of enzymes and of corresponding mt plasmids. The putative amino acidic sequence of pUG1 shares a high degree of similarity with those of two Neurospora intermedia plasmids, Fiji and LaBelle. They are characterised by a specific signature in the motif C typical of the family B DNA polymerases, TTD instead of DTD, that presumably is a typical motif of this subgroup of enzymes. The three plasmids also share a large number of amino acids typical of the protein primed DNA polymerases found in linear mt plasmids, linear bacteriophages and viruses. Finally pUG1, Fiji and LaBelle show a similar size and structure of their genomes confirming their belonging to a distinct group. In the context of the evolutionary and population biology of the pUG1 plasmids, a survey of a world collection of 154 strains of C. parasitica was conducted, the isolates were screened by specific PCR and amplified DNAs from some representative strains were sequenced and analysed.
111 The scooter transposons are not unique to Schizophyllum commune. Cynthia L. St. Hilaire, Thomas J. Fowler, and Carlene A. Raper. Department of Microbiology and Molecular Genetics, University of Vermont, Burlington VT 05405 USA.
Scooter transposons are DNA-mediated transposons first identified in the homobasidiomycete Schizophyllum commune. Two copies of scooter previously characterized, scooter-1 and scooter-2, are 91% identical over their ~ 650 bp. They are nonautonomous elements; an autonomous element has not yet been identified. Scooter elements can transpose and their insertions have led to gene disruptions. Depending on the strain of S. commune tested, anywhere from 3 to 25 restriction fragments have been identified with a scooter probe on genomic Southern blots. We were curious as to whether scooter was confined to this one species, or if other mushroom fungi also harbor scooter elements. Genomic DNA from several mushroom species was probed with scooter at low stringency in a Southern blot analysis. Hybridization with scooter was evident in several of other mushroom fungi, including Agaricus bisporus, Pleurotus ostreatus, and Lentinus edodes, but the hybridization signal is much weaker for these other fungi than for S. commune. Different varieties of A. bisporus, the button and Portobella varieties, have similar hybridization patterns to suggest these scooter-related sequences may not transpose in A. bisporus. We are attempting to isolate some of these scooter-related sequences with PCR to ascertain their relationship to the S.commune scooter elements.
112 AFLP diversity of Cephalosporium maydis in Egypt. A. A. Saleh1, K. A. Zeller1, E. M. El-Assiuty2 and J. F. Leslie1. 1Department of Plant Pathology, Kansas State University, Manhattan, KS 66506-5502 2Maize Section, Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt
We characterized more than 868 isolates of Cephalosporium maydis collected from 14 governates in Egypt with AFLP markers. These governates included seven located in lower Egypt and seven located in upper Egypt. The four primer-pair combinations resulted in 25 polymorphic markers from total of 68. UPGMA clustering analysis results in four groups (lineages) that are not uniformly distributed throughout the country. Lineage four was not recovered from any of the seven upper Egypt governates whereas the other three lineages were found in both upper and lower Egypt. Lineage four was the most divergent group. In some locations, one lineage dominated (up to 98%) and some fields were colonized only by isolates that belonged to the same clone. Differences in climate or maize varieties planted might be sufficient to explain the unusual distribution of lineage four.
113 Cloning and analysis of the mating type genes from the barley pathogen Septoria passerinii. Stephen B. Goodwin1, Cees Waalwijk2, Gerrit H. J. Kema 2 and Jessica R. Cavaletto1. 1 USDA-ARS, Department of Botany and Plant Pathology, 1155 Lilly Hall, Purdue University, West Lafayette, IN 47907-1155; 2 Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands
Septoria passerinii causes speckled leaf blotch of barley. No teleomorph has been found and the pathogen is assumed to reproduce through asexual pycnidiospores. Recent phylogenetic analyses have shown that S. passerinii is closely related to the wheat pathogen Mycosphaerella graminicola, a species with regular sexual reproduction. Analyses of genetic variation within populations of S. passerinii revealed a high level of genotypic diversity, suggesting the possibility of sexual reproduction in nature. To test the hypothesis that S. passerinii is capable of sexual reproduction, mating type clones from M. graminicola were used as probes in Southern analyses. A 4 kb band was present in some isolates of S. passerinii when probed with a clone containing the HMG box from M. graminicola. This 4176 bp band contained a complete HMG box-like idiomorph of 2899 bp from S. passerinii, including sequence at both flanking regions. An open reading frame (ORF) of 101 amino acids had high similarity to the HMG box region of M. graminicola and other fungi. PCR primers were designed to amplify the other mating type idiomorph from isolates that did not hybridize to the HMG box probe. Sequencing of a 3637 bp PCR product revealed an alpha protein-containing idiomorph of 3050 bp. This clone contained an ORF of 345 amino acids with high similarity to alpha mating-type proteins from other fungi. Primers for multiplex PCR were designed to test the mating types from field isolates. This technique revealed that both mating types were present in the same fields in Minnesota and North Dakota. Therefore, this pathogen may have the potential for sexual reproduction in nature, which could explain the high levels of genotypic diversity observed in barley fields in the north central U.S.
114 Evidence for domestication of the fungal symbionts of leafcutter ants. Stephen A. Rehner. Insect Biocontrol Laboratory, ARS, USDA. Beltsville, Maryland, USA.
Whether the fungi cultivated by leafcutter ants are truly domesticated has posed an enigma for over 100 years. Analyses of genetic variation among leafcutter cultivars at local and geographic scales were conducted to see if their genetic structure departed from a null expectation of a recombinatorial interbreeding population structure. AFLP variation among cultivars from central Panama revealed a hierarchical genetic structure consistent with the traditional view that the fungi are clonal. However, mycelial incompatibility between pairs of genetically different cultivars provide evidence that these fungi both retain and exercise the ability for sexual reproduction. Phylogeographic analysis of cultivars demonstrated that alleles of different loci are concordantly partitioned among geographically structured. These data support the conclusion that the leafcutter fungi comprise a series of inbreeding cultivar lines that exist predominantly, if not exclusively, in association with their ant hosts.
115 Evolutionary relationships of kinesins in fungi. Conrad L. Schoch, B. Gillian Turgeon, Olen C. Yoder and James R. Aist. Department of Plant Pathology, Cornell University, Ithaca, New York
Kinesins are mechanochemical proteins able to move cargo along microtubules by ATP hydrolysis. Together with dyneins and myosins they are motor proteins, involved in a number of vital cellular processes such as organelle transport, chromosome segregation and cytokinesis. These proteins have been isolated from a wide range of organisms ranging from humans to yeast and their evolutionary relationships compared and analysed in previous studies. Among fungi valuable insights into function have been gained from functional analysis in organisms such as the ascomycetous yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the filamentous ascomycetes Aspergillus nidulans, Neurospora crassa and Nectria haematococca, as well as the basidiomycete Ustilago maydis. A computer generated search with available genomic sequences of the plant pathogenic fungi Fusarium graminearum, Cochliobolus heterostrophus and Botrytis cinerea has indicated that the numbers of putative genes containing kinesin motor domains in each fungus is comparable to those already known from S. cerevisiae. A phylogenetic analysis is presented.
116 The use of molecular phylogenies for estimation of fungal diversity. Jean-Marc Moncalvo and Rytas Vilgalys. Duke University Department of Biology, Durham NC 27708
Classic estimates of biological diversity use species (or genera) as units of measurement. The development of large-scale molecular phylogenies provides an unique opportunity to estimate biological diversity at the genetic level by using the branching order and branch lengths from phylogenetic trees. Both among-taxa and among-area phylogenetic diversity can be estimated using a "phylogenetic index of diversity" (PD). We will contrast PD with classic estimates of biological diversity in various groups of mushrooms for which extensive geographic sampling and molecular phylogenies are available, including genera Amanita, Pleurotus, Lentinula, and Ganoderma. There are at least two advantages of PD over traditional measures of estimating biological diversity: 1) PD does not require a priori knowledge of taxonomic circumscription (which is generally subjective in fungi), and 2) PD takes into account genetic distances, therefore emphasizes genetic breadth when estimating diversity.
117 Molecular population genetics of arbuscular
mycorrhizal fungi: Are they clonal or recombining? Teresa E. Pawlowska and John
W. Taylor. University of California, Plant & Microbial Biology, Berkeley, CA 94720,
Arbuscular mycorrhizal fungi (Glomales) form symbioses with the majority of plant species, including many crops. They facilitate plant mineral nutrition and confer tolerance to pathogens, drought, salinity and metal toxicity. Glomales are obligate biotrophs with no evidence of sexual reproduction. The location of genetic variation in their multinucleate hyphae is unknown. It may be in each nucleus (homokaryotic) or among several nuclei (heterokaryotic). Before we can challenge the hypothesis that Glomales are clonal, we need to establish whether they are homo- or heterokaryotic. As a model we are using Glomus etunicatum, a ubiquitous species that can be easily sampled from nature and cultured in vitro. To obtain a representation of its wild populations, we collected soil samples from Berkeley, CA, and St. Paul, MN. Spores recovered from greenhouse trap cultures were used to initiate single spore cultures of G. etunicatum in association with excised Ri T-DNA transformed carrot roots. To search for polymorphic markers in single copy genes in addition to rDNA arrays, and to enable PCR amplification of multiple loci from individual Glomus spores, we optimized a strategy for global amplification of spore DNA. Putative genes encoding a DEAD box protein, a catalytic subunit of DNA polymerase alpha, a protein with a putative leucine zipper DNA-binding element, and an ADP- ribosylation factor-like protein were identified as genetic markers. To test for homo- vs. heterokaryosis, we are analyzing progeny spores from single spore cultures for variation at the polymorphic loci. If all the progeny are identical, there would be no evidence for heterokaryosis, and we would be able to proceed to testing of reproductive mode of Glomus in nature.
118 The quest for nucleotide polymorphism in Aspergillus fumigatus; implications for a phylogenetic definition of the species. J.L. Platt1, D.M. Geiser2, & J.W. Taylor1. 1University of California, Berkeley, CA USA. 2The Pennsylvania State University, University Park, PA USA.
Phylogenetic analyses of several human pathogenic fungi have shown that species traditionally defined on the basis of morphology may actually represent more than one genetically isolated species. As a first objective in our effort to understand the reproductive mode and population genetics of Aspergillus fumigatus, we are re-examining the species boundary in this asexual pathogen. We conducted a preliminary screening for polymorphic sites in 28 loci of both clinical and environmental isolates. Phylogenetic analyses of nucleotide sequence data from eight loci in exemplar isolates that include two varieties of A. fumigatus(var. acolumnaris and var. ellipticus) were used to infer existence of genetic isolation. While sequences of catalase (catB) and polyketide synthase (pksP) showed some nucleotide variation within all isolates, the remaining loci (chsG, alp1, mep,and gp55)exhibited very little nucleotide variation even between varieties. A comparison of the resulting gene genealogies did not result in any patterns that would be consistent with cryptic speciation and is counter to other studies which indicate that cryptic speciation may be relatively common in fungi. We are now pursuing several other potential sources of polymorphic nucleotide sites, including uncharacterized loci from BAC end sequences, intergenic regions, and microsatellite flanking sequences. Phylogenetic analyses of more variable markers will be used to further test the traditional morphology-based species concept of A. fumigatus.
119 Population genetics of Ustilago maydis as determined by RFLP and allelic variation and the b mating type locus. James R. Garton, Georgiana May and Christine E Ramos. University of Minnesota, Plant Biology, St. Paul, MN
We investigated the population genetic structure of Ustilago maydis, with particular emphasis on migration levels between geographically distant populations. We examined genetic diversity in this organism by the use of 13 RFLP probes and characterization of allelic diversity at the b mating type locus. We performed analyses on 276 haploid individuals sampled from seven subpopulations. Six of these populations were in the US while one was from Uruguay. Low to moderate genetic variability was observed at the neutral loci in all US populations (pairwise Fst ranges from 0.1 to 0.25), while substantial variability was found between US populations and Uruguay (average Fst of 0.4). The variability detected at the b-locus was universally low (Fst of 0.1). Calculation of migration levels by Slatkin's private allele method revealed moderate gene flow in the US populations (Nm=3), and no gene flow between the US and South America (Nm=0.3). Mantel tests revealed no correlation between genetic and geographic distance for populations in the US (correlation coefficient of -0.006), and moderate correlation between the US and Uruguayan population (correlation coefficient of 0.6). The low level of genetic diversity observed at the mating type locus is the result of frequency dependent selection maintaining a high level of variation at this locus. It is our conclusion that the results from the neutral markers indicate that this fungus demonstrates moderate levels of short range gene flow while virtually no migrants are exchanged over long distances (South America to the US).
120 Clone size, fine-scale population structure, and phylogenetic species in the ectomycorrhizal false-truffle Rhizopogon vinicolor complex. Annette M. Kretzer1, Lisa C. Grubisha2, Randy Molina3, and Joseph W. Spatafora1. 1Dept. of Botany & Plant Pathology, Oregon State University, Corvallis, OR, USA. 2Dept. of Plant & Microbial Biology, University of California, Berkeley, CA, USA. 3US Forest Service, PNW Research Station, Corvallis, OR, USA.
A population genetic study was initiated to study the population dynamics of the ectomycorrhizal false-truffle Rhizopogon vinicolor. R. vinicolor is host specific with Pseudotsuga menziesii and produces hypogeous sporocarps and nonforcibly discharged spores that are primarily dispersed through small mammal mycophagy. R. vinicolor was chosen as a model system for studying population biology of truffle-forming ectomycorrhizal fungi for numerous reasons including host specificity, common occurrence in nature, and ease of sampling. This last factor is attributed to the fact that R. vinicolor is the only false-truffle reported to produce tuberculate mycorrhizae, which consist of clusters of ectomycorrhizal roottips encased in a peridium. These tuberculate mycorrhizae are relatively easy to sample in nature and are more widely distributed than sporocarps. We developed numerous R. vinicolor-specific, single-copy microsatellite markers to address several questions including clone size and distribution, fine scale population fragmentation, and gene flow in the context of isolation by distance and barriers. Our initial results supported that clone size ranged from less than 5 meters to approximately 15 meters in diameter. More exhaustive sampling and analyses revealed that two sympatrically distributed Rhizopogon species produce tuberculate mycorrhizae, which we distinguish here as R. vinicolor s.s. and R. cf. vinicolor. These results were corroborated by the sampling of sporocarps including type specimens, the lack of shared alleles across microsatellite loci, and through phylogenetic analysis of the ITS rDNA. We will also present preliminary data on fine-scale population structure at the water shed level and relative rates of inbreeding.
121 Combined genotyping methods for indoor moulds. James Scott1 and Wieland Meyer2. 1University of Toronto, Botany, Toronto, ON. Canada. 2University of Sydney, Westmead Hospital, Westmead NSW Australia.
Penicillium chrysogenum is one of the most common microfungi isolated from indoor environments. Moreover, this species is of considerable industrial importance as the principal producer of the antibiotic, penicillin. This study used two genotyping methods, heteroduplex mobility assay (HMA) and PCR fingerprinting to examine the extent of clonality within this species.
Thirty-eight isolates of P. chrysogenum were obtained from broadloom dust representing 27 houses from Wallaceburg, Ontario, Canada and neighboring rural areas. One isolate each of P. polonicum and P. thomii were used as outgroups. Allelic variability was assessed using HMA in PCR-amplified polymorphic genetic loci including three regions spanning introns in the conserved structural or metabolic genes acetyl-Coenzyme-A synthase (acuA), beta- tubulin (benA) and thioredoxin reductase (trxB), as well as the internal transcribed spacer region (ITS) of the nuclear ribosomal RNA gene. The identity of alleles was confirmed by sequencing. The same panel of isolates was genotyped by PCR fingerprinting using single-primer PCR with the minisatellite-specific core sequence of the wild-type phage M13, and the microsatellite-specific motif (GACA)4. In addition, 2-primer PCR was conducted using the two RAPD primers, 5SOR and MYC1.
HMA revealed three alleles at each locus that assorted as 5 multilocus haplotypes. All alleles showed strong association and no incompatibilities were observed, indicating strict clonality. Phylogenetic analysis of sequences of the combined loci revealed three well-supported clades. Results of PCR fingerprinting similarly identified three primary lineages, however, these methods provided considerably better resolution within these clades. The taxonomic implications of these results will be discussed.
122 Evolution of spacer regions of nuclear ribosomal multigene family in Fusarium culmorum. P. K. Mishra, R. T. V. Fox and A. Culham. School of Plant Sciences, The University of Reading, Whiteknights, RG6 6AS, U.K.
The genus Fusarium contains many agronomically and clinically important species. Fusarium culmorum is of particular importance due to its significance not only in plant pathology but also in mycotoxicology. In this study, we analyze the evolutionary dynamics of the spacer regions of multigene family of nuclear ribosomal DNA. The sequence data analyzed deruve from the internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of seventy five strains of Fusarium culmorum representing different hosts and geographical origins. Our extensive molecular analysis of sequence data reveals a contrasting pattern of evolution in ITS and IGS regions. Multiple examples of apparent gene duplication, substitutions and indel events were observed in IGS region. In contrast, it seems that ITS region is completely homogenized among the Fusarium culmorum strains exhibiting only 0.35% sequence divergence. Thus, it seems apparent that a different mode of concerted evolution is operating in the spacer regions of nuclear ribosomal DNA in Fusarium culmorum. The possible models of homogenization have been discussed.
123 Analysis of a eukaryotic microbial mat community across environmental gradients in a thermal, acidic stream. Kathy B. Sheehan, Michael J. Ferris, and Joan M. Henson. Department of Microbiology and the Thermal Biology Institute, Montana State University, Bozeman, MT
Nymph Creek in Yellowstone National Park is a natural laboratory for understanding eukaryotic microbial genetic diversity, ecophysiology, and behavior. The thermal(50oC), acidic(pH 2.7) water creates stable environmental gradients in temperature, pH, and light over which changes in microbial populations are being monitored using microscopic methods and rRNA sequencing during diurnal and seasonal periods. The mat is primarily composed of the red alga, Cyanidium caldarium, the most thermophilic alga known, and the thermophilic, filamentous fungus, Dactylaria constricta var. gallopava. The distributions of fungal and algal 18S rRNA genotypes provide information on how genotypes correlate with ecological niches. Of particular interest is the interaction between C. caldarium and D. constricta, which could represent a primitive symbiotic ancestor to modern-day lichens. In addition, we are studying whether the alga provides nutrients for the fungus, whether the fungus parasitizes the alga, whether fungal hyphae provide a matrix that anchors the unicellular alga to form a mat and whether fungal melanins protect the alga from the harmful effects of intense light at high elevations. This project is supported by a National Science Foundation Microbial Observatory grant and by the Thermal Biology Institute at Montana State University.
124 Human disturbance fosters hybridization in the fungal tree pathogen Heterobasidion. M. Garbelotto, W. Otrosina, and I. Chapela. University of California, ESPM-Ecosysytem Sciences, Berkeley, CA, USA
Hybrids of two host-specific taxa in the pathogenic fungal genus Heterobasidion were less virulent than parental strains on either of the adaptive hosts in greenhouse experiments. However, we did not find a hybrid disadvantage on stumps and on the dual-host Sitka spruce, representing alternate non-selective infection courts. In California, non-selective courts have been massively produced only in the recent past, due to logging and fire-control practices. We provide evidence that these two management practices have lead to conditions conducive to hybridization and interspecifiec gene flow. Based on discordant genealogies we show that interspecific gene flow has also occurred in the past between the two North American taxa of the genus Heterobasidion 39 Using a Shot-Gun Genomic Microarray to Probe Pathogenesis in Histoplasma capsulatum. Margareta Andersson, Adam Bahrami, M. Paige Nittler, and Anita Sil The goal of our work is to identify regulators of pathogenesis in the dimorphic fungus Histoplasma capsulatum, a primary pathogen that causes severe disease in immunocompromised patients. H. capsulatum grows in a mycelial form in soil. Conidia or hyphal fragments are inhaled by the host; once inside the host, the cells undergo a morphogenetic switch and grow as a budding yeast form which parasitizes macrophages. The molecular regulators of this dimorphic switch, which is thought to be essential for establishment of infection, are unknown. Similarly, how H. capsulatum is able to escape killing by macrophages and colonize the phagolysosome, an intracellular niche that is normally hostile to microbes, is a mystery. We, in collaboration with Lena Hwang and Jasper Rine at UCBerkeley, have designed a powerful new tool that can be used to expeditiously identify key regulatory proteins in the life cycle of this dimorphic pathogen. We have built a 9600- element H. capsulatum DNA microarray and have used it successfully to identify new yeast-specific and mycelial-specific genes whose differential expression has been confirmed by Northern analysis. We are now poised to use genomics as a high through-put method to dissect key steps in the infectious process such as colonization of macrophages. We will present preliminary data pertinent to the gene expression profile of H. capsulatum that has colonized macrophages.
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