Database : the journal of biological databases and curation, 2014
DNA phylogenetic comparisons have shown that morphology-based species recognition often underesti... more DNA phylogenetic comparisons have shown that morphology-based species recognition often underestimates fungal diversity. Therefore, the need for accurate DNA sequence data, tied to both correct taxonomic names and clearly annotated specimen data, has never been greater. Furthermore, the growing number of molecular ecology and microbiome projects using high-throughput sequencing require fast and effective methods for en masse species assignments. In this article, we focus on selecting and re-annotating a set of marker reference sequences that represent each currently accepted order of Fungi. The particular focus is on sequences from the internal transcribed spacer region in the nuclear ribosomal cistron, derived from type specimens and/or ex-type cultures. Re-annotated and verified sequences were deposited in a curated public database at the National Center for Biotechnology Information (NCBI), namely the RefSeq Targeted Loci (RTL) database, and will be visible during routine sequenc...
We report the annotated draft genome sequence of Lichtheimia ramosa (JMRC FSU:6197). It has been ... more We report the annotated draft genome sequence of Lichtheimia ramosa (JMRC FSU:6197). It has been reported to be a causative organism of mucormycosis, a rare but rapidly progressive infection in immunocompromised humans. The functionally annotated genomic sequence consists of 74 scaffolds with a total number of 11,510 genes.
... 2006). The morphological taxonomy of zygomycetous fungi was extensively investigated during t... more ... 2006). The morphological taxonomy of zygomycetous fungi was extensively investigated during the last 50 years by some experts (Benjamin 1959, 1966, 1979; Hesseltine and Ellis 1973; O'Donnell 1979; Benny 1982; Lichtwardt 1986; Humber 1989; Benny et al. ...
Lichtheimia species are the second most important cause of mucormycosis in Europe. To provide bro... more Lichtheimia species are the second most important cause of mucormycosis in Europe. To provide broader insights into the molecular basis of the pathogenicity-associated traits of the basal Mucorales, we report the full genome sequence of L. corymbifera and compared it to the genome of Rhizopus oryzae, the most common cause of mucormycosis worldwide. The genome assembly encompasses 33.6 MB and 12,379 protein-coding genes. This study reveals four major differences of the L. corymbifera genome to R. oryzae: (i) the presence of an highly elevated number of gene duplications which are unlike R. oryzae not due to whole genome duplication (WGD), (ii) despite the relatively high incidence of introns, alternative splicing (AS) is not frequently observed for the generation of paralogs and in response to stress, (iii) the content of repetitive elements is strikingly low (<5%), (iv) L. corymbifera is typically haploid. Novel virulence factors were identified which may be involved in the regul...
Two filamentous fungi with different phenotypes were isolated from crushed healthy spores or perf... more Two filamentous fungi with different phenotypes were isolated from crushed healthy spores or perforated dead spores of the arbuscular mycorrhizal fungus (AMF) Scutellospora castanea. Based on comparative se- quence analysis of 5.8S ribosomal DNA and internal transcribed spacer fragments, one isolate, obtained from perforated dead spores only, was assigned to the genus Nectria, and the second, obtained from both healthy
Database : the journal of biological databases and curation, 2014
DNA phylogenetic comparisons have shown that morphology-based species recognition often underesti... more DNA phylogenetic comparisons have shown that morphology-based species recognition often underestimates fungal diversity. Therefore, the need for accurate DNA sequence data, tied to both correct taxonomic names and clearly annotated specimen data, has never been greater. Furthermore, the growing number of molecular ecology and microbiome projects using high-throughput sequencing require fast and effective methods for en masse species assignments. In this article, we focus on selecting and re-annotating a set of marker reference sequences that represent each currently accepted order of Fungi. The particular focus is on sequences from the internal transcribed spacer region in the nuclear ribosomal cistron, derived from type specimens and/or ex-type cultures. Re-annotated and verified sequences were deposited in a curated public database at the National Center for Biotechnology Information (NCBI), namely the RefSeq Targeted Loci (RTL) database, and will be visible during routine sequenc...
Anaerobic fungi are phylogenetically unique and form a separate group, the Neocallimastigomycota,... more Anaerobic fungi are phylogenetically unique and form a separate group, the Neocallimastigomycota, among the chitinous fungi. Until now six genera are described within that phylum, namely the monocentric genera Neocallimastix, Caecomyces and Piromyces as well as the polycentric genera Anaeromyces, Cyllamyces and Orpinomyces. This chapter gives a brief survey of the fascinating world of anaerobic rumen fungi, their phylogeny, and identification. The golden standards of molecular identification as well as promising alternatives will be discussed.
The rice seedling blight fungus Rhizopus microsporus harbors endosymbiotic Burkholderia sp. for t... more The rice seedling blight fungus Rhizopus microsporus harbors endosymbiotic Burkholderia sp. for the production of the virulence factor, the antimitotic agent rhizoxin. Since the toxin highly efficiently blocks mitosis in most eukaryotes, it remained elusive how self-resistance emerged in the fungal host. In this study, rhizoxin sensitivity was systematically correlated with the nature of b-tubulin sequences in the kingdom Fungi. A total of 49 new b-tubulin sequences were generated for representative species of Ascomycota, Basidiomycota and Zygomycota. Rhizoxin sensitivity assays revealed two further amino acids at position 100 (Ser-100 and Ala-100), in addition to the known Ile-100 and Val-100, which convey rhizoxin resistance. All sensitive strains feature Asn-100. This hot spot was verified by modeling studies, which support the finding that rhizoxin preferentially interacts with the tubulin molecule in a cavity near position 100. Ancestral character state reconstructions conducted in a Bayesian framework suggest that rhizoxin sensitivity represents the ancestral character state in fungi, and that evolution of rhizoxin resistance took place in the ancestor of extant resistant Zygomycota. These findings support a model according to which endosymbiosis became possible through a parasitism-mutualism shift in insensitive fungi.
Among genes coding for proteins with basic structural functions in all eukaryotes, the highly con... more Among genes coding for proteins with basic structural functions in all eukaryotes, the highly conserved and functionally essential gene for βtubulin is receiving increasing attention in the reconstruction of phylogenies within a broad organismic range. We therefore constructed a set of twelve universally applicable primers that allow reliable amplification of β-tubulin genes among all major eukaryotic kingdoms including fungi (Fungi), animals (Animalia) and green plants (Planta). For primer design, the amino acid sequences of 35 β-tubulin genes from Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, Animalia, Oophyta and Planta were aligned and used for the definition of four well-conserved regions. These are suitable priming sites in PCR amplification experiments. Out of these amino acid regions twelve primers were designed which initiate especially the amplification of fungal β-tubulin genes. In four pairwise primer applications gene fragments of up to 1500 bp in size could be isolated, which comprise nearly complete β-tubulin genes from twelve species representative of the Fungi. The sequences of seven β-tubulin fragments were obtained from Allomyces moniliformis, A. neomoniliformis, Blastocladiella britannica, Chytridium confervae, Mortierella isabellina and Trametes versicolor, respectively. Reliable amplification of β-tubulin over a broad spectrum of organisms provides a strong basis for the establishment of both deep-level phylogenies and studies of complex species groups based on β-tubulin gene trees.
The dothideomycetous fungus Leptosphaeria maculans comprises a complex of species diVering in spe... more The dothideomycetous fungus Leptosphaeria maculans comprises a complex of species diVering in speciWcity and pathogenicity on Brassica napus. Twenty-eight isolates were investigated and compared to 20 other species of the Pleosporales order. Sequences of the mating type MAT1-2 (23), fragments of actin (48) and -tubulin (45) genes were determined and used for phylogenetic analyses inferred by maximum parsimony, distance, maximum likelihood, and Bayesian approaches. These diVerent approaches using single genes essentially conWrmed Wndings using concatanated sequences. L. maculans formed a monophyletic group separate from Leptosphaeria biglobosa. The L. biglobosa clade encompasses Wve sub-clades; this Wnding is consistent with classiWcation made previously on the basis of internal-transcribed sequences of the ribosomal DNA repeat. The propensity for purifying and neutral evolution of the three genes was determined using sliding window analysis, a technique not previously applied to genes of Wlamentous fungi. For members of the L. maculans species complex, this approach showed that in comparison to actin and -tubulin, exonic sequences of MAT1-2 were more diverse and appeared to evolve at a faster rate. However, diVerent regions of MAT1-2 displayed diVerent degrees of sequence conservation. The more conserved upstream region (including the High Mobility Group domain) may be better suited for interspecies diVerentiation, while the more diverse downstream region is more appropriate for intraspecies comparisons.
The gene for actin as a highly conserved and functionally essential genetic element is developing... more The gene for actin as a highly conserved and functionally essential genetic element is developing into a major tool for phylogenetic analysis within a broad organismic range. We therefore propose a set of universally applicable primers that allow reliable amplification of actin genes. For primer construction the amino acid sequences of 57 actin genes comprising fungi, animals, plants and protists were analysed, aligned and used for the definition of six well-conserved regions which are suitable as priming sites in PCR amplification experiments. Ten primers were designed for specific in vitro amplification of actin gene fragments from a wide range of microorganisms. The corresponding gene fragments provide a strong basis to isolate nearly complete actin genes for further molecular characterization and for establishing phylogenies based on actin gene trees.
The randomly amplified polymorphic DNA technique (RAPD) is a modification of peR that uses short,... more The randomly amplified polymorphic DNA technique (RAPD) is a modification of peR that uses short, arbitrarily generated single primers to amplify genomic DNA. Amplified DNA-fragments are often polymorphic and can be used as individual, population-or species-specific markers. Because the RAPD technique requires a very high degree of reproducibility at the instrumentation level and with regard to buffer conditions, we propose to synthesize highly specific conventional PCR primers, the sequence of which is based on the primary diagnostic RAPD bands. In this communication we present a fast and convenient experimental strategy for converting the non-stringent RAPD conditions with their low annealing temperatures to stringent PCR conditions. Diagnostic RAPD bands were sequenced by a combination of chemical (Gilbert/ Maxam) and chain termination (Sanger) techniques. Based on this sequence information, highly specific oligonucleotide primers were synthesized. The value of this approach was demonstrated for the molecular diagnosis of the important rape seed (Brassica napus) pathogen Leptosphaeria maculans.
47 Polish isolates of the blackleg fungus Leptosphaeria maculans (Phoma lingam) were compared wit... more 47 Polish isolates of the blackleg fungus Leptosphaeria maculans (Phoma lingam) were compared with eight well-defined reference strains from Germany, France, Denmark, Australia and one Polish isolate of Phoma nigrificans. The isolates were tested (i) for growth characteristics, (ii) for their ability to form sirodesmins, (iii) for cellulolytic enzymes, and (iv) for pathotype-differentiating molecular markers generated by RAPD-PCR, PCR analysis with pathotype-specific primer pairs and PFGE.
Database : the journal of biological databases and curation, 2014
DNA phylogenetic comparisons have shown that morphology-based species recognition often underesti... more DNA phylogenetic comparisons have shown that morphology-based species recognition often underestimates fungal diversity. Therefore, the need for accurate DNA sequence data, tied to both correct taxonomic names and clearly annotated specimen data, has never been greater. Furthermore, the growing number of molecular ecology and microbiome projects using high-throughput sequencing require fast and effective methods for en masse species assignments. In this article, we focus on selecting and re-annotating a set of marker reference sequences that represent each currently accepted order of Fungi. The particular focus is on sequences from the internal transcribed spacer region in the nuclear ribosomal cistron, derived from type specimens and/or ex-type cultures. Re-annotated and verified sequences were deposited in a curated public database at the National Center for Biotechnology Information (NCBI), namely the RefSeq Targeted Loci (RTL) database, and will be visible during routine sequenc...
We report the annotated draft genome sequence of Lichtheimia ramosa (JMRC FSU:6197). It has been ... more We report the annotated draft genome sequence of Lichtheimia ramosa (JMRC FSU:6197). It has been reported to be a causative organism of mucormycosis, a rare but rapidly progressive infection in immunocompromised humans. The functionally annotated genomic sequence consists of 74 scaffolds with a total number of 11,510 genes.
... 2006). The morphological taxonomy of zygomycetous fungi was extensively investigated during t... more ... 2006). The morphological taxonomy of zygomycetous fungi was extensively investigated during the last 50 years by some experts (Benjamin 1959, 1966, 1979; Hesseltine and Ellis 1973; O&#x27;Donnell 1979; Benny 1982; Lichtwardt 1986; Humber 1989; Benny et al. ...
Lichtheimia species are the second most important cause of mucormycosis in Europe. To provide bro... more Lichtheimia species are the second most important cause of mucormycosis in Europe. To provide broader insights into the molecular basis of the pathogenicity-associated traits of the basal Mucorales, we report the full genome sequence of L. corymbifera and compared it to the genome of Rhizopus oryzae, the most common cause of mucormycosis worldwide. The genome assembly encompasses 33.6 MB and 12,379 protein-coding genes. This study reveals four major differences of the L. corymbifera genome to R. oryzae: (i) the presence of an highly elevated number of gene duplications which are unlike R. oryzae not due to whole genome duplication (WGD), (ii) despite the relatively high incidence of introns, alternative splicing (AS) is not frequently observed for the generation of paralogs and in response to stress, (iii) the content of repetitive elements is strikingly low (<5%), (iv) L. corymbifera is typically haploid. Novel virulence factors were identified which may be involved in the regul...
Two filamentous fungi with different phenotypes were isolated from crushed healthy spores or perf... more Two filamentous fungi with different phenotypes were isolated from crushed healthy spores or perforated dead spores of the arbuscular mycorrhizal fungus (AMF) Scutellospora castanea. Based on comparative se- quence analysis of 5.8S ribosomal DNA and internal transcribed spacer fragments, one isolate, obtained from perforated dead spores only, was assigned to the genus Nectria, and the second, obtained from both healthy
Database : the journal of biological databases and curation, 2014
DNA phylogenetic comparisons have shown that morphology-based species recognition often underesti... more DNA phylogenetic comparisons have shown that morphology-based species recognition often underestimates fungal diversity. Therefore, the need for accurate DNA sequence data, tied to both correct taxonomic names and clearly annotated specimen data, has never been greater. Furthermore, the growing number of molecular ecology and microbiome projects using high-throughput sequencing require fast and effective methods for en masse species assignments. In this article, we focus on selecting and re-annotating a set of marker reference sequences that represent each currently accepted order of Fungi. The particular focus is on sequences from the internal transcribed spacer region in the nuclear ribosomal cistron, derived from type specimens and/or ex-type cultures. Re-annotated and verified sequences were deposited in a curated public database at the National Center for Biotechnology Information (NCBI), namely the RefSeq Targeted Loci (RTL) database, and will be visible during routine sequenc...
Anaerobic fungi are phylogenetically unique and form a separate group, the Neocallimastigomycota,... more Anaerobic fungi are phylogenetically unique and form a separate group, the Neocallimastigomycota, among the chitinous fungi. Until now six genera are described within that phylum, namely the monocentric genera Neocallimastix, Caecomyces and Piromyces as well as the polycentric genera Anaeromyces, Cyllamyces and Orpinomyces. This chapter gives a brief survey of the fascinating world of anaerobic rumen fungi, their phylogeny, and identification. The golden standards of molecular identification as well as promising alternatives will be discussed.
The rice seedling blight fungus Rhizopus microsporus harbors endosymbiotic Burkholderia sp. for t... more The rice seedling blight fungus Rhizopus microsporus harbors endosymbiotic Burkholderia sp. for the production of the virulence factor, the antimitotic agent rhizoxin. Since the toxin highly efficiently blocks mitosis in most eukaryotes, it remained elusive how self-resistance emerged in the fungal host. In this study, rhizoxin sensitivity was systematically correlated with the nature of b-tubulin sequences in the kingdom Fungi. A total of 49 new b-tubulin sequences were generated for representative species of Ascomycota, Basidiomycota and Zygomycota. Rhizoxin sensitivity assays revealed two further amino acids at position 100 (Ser-100 and Ala-100), in addition to the known Ile-100 and Val-100, which convey rhizoxin resistance. All sensitive strains feature Asn-100. This hot spot was verified by modeling studies, which support the finding that rhizoxin preferentially interacts with the tubulin molecule in a cavity near position 100. Ancestral character state reconstructions conducted in a Bayesian framework suggest that rhizoxin sensitivity represents the ancestral character state in fungi, and that evolution of rhizoxin resistance took place in the ancestor of extant resistant Zygomycota. These findings support a model according to which endosymbiosis became possible through a parasitism-mutualism shift in insensitive fungi.
Among genes coding for proteins with basic structural functions in all eukaryotes, the highly con... more Among genes coding for proteins with basic structural functions in all eukaryotes, the highly conserved and functionally essential gene for βtubulin is receiving increasing attention in the reconstruction of phylogenies within a broad organismic range. We therefore constructed a set of twelve universally applicable primers that allow reliable amplification of β-tubulin genes among all major eukaryotic kingdoms including fungi (Fungi), animals (Animalia) and green plants (Planta). For primer design, the amino acid sequences of 35 β-tubulin genes from Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, Animalia, Oophyta and Planta were aligned and used for the definition of four well-conserved regions. These are suitable priming sites in PCR amplification experiments. Out of these amino acid regions twelve primers were designed which initiate especially the amplification of fungal β-tubulin genes. In four pairwise primer applications gene fragments of up to 1500 bp in size could be isolated, which comprise nearly complete β-tubulin genes from twelve species representative of the Fungi. The sequences of seven β-tubulin fragments were obtained from Allomyces moniliformis, A. neomoniliformis, Blastocladiella britannica, Chytridium confervae, Mortierella isabellina and Trametes versicolor, respectively. Reliable amplification of β-tubulin over a broad spectrum of organisms provides a strong basis for the establishment of both deep-level phylogenies and studies of complex species groups based on β-tubulin gene trees.
The dothideomycetous fungus Leptosphaeria maculans comprises a complex of species diVering in spe... more The dothideomycetous fungus Leptosphaeria maculans comprises a complex of species diVering in speciWcity and pathogenicity on Brassica napus. Twenty-eight isolates were investigated and compared to 20 other species of the Pleosporales order. Sequences of the mating type MAT1-2 (23), fragments of actin (48) and -tubulin (45) genes were determined and used for phylogenetic analyses inferred by maximum parsimony, distance, maximum likelihood, and Bayesian approaches. These diVerent approaches using single genes essentially conWrmed Wndings using concatanated sequences. L. maculans formed a monophyletic group separate from Leptosphaeria biglobosa. The L. biglobosa clade encompasses Wve sub-clades; this Wnding is consistent with classiWcation made previously on the basis of internal-transcribed sequences of the ribosomal DNA repeat. The propensity for purifying and neutral evolution of the three genes was determined using sliding window analysis, a technique not previously applied to genes of Wlamentous fungi. For members of the L. maculans species complex, this approach showed that in comparison to actin and -tubulin, exonic sequences of MAT1-2 were more diverse and appeared to evolve at a faster rate. However, diVerent regions of MAT1-2 displayed diVerent degrees of sequence conservation. The more conserved upstream region (including the High Mobility Group domain) may be better suited for interspecies diVerentiation, while the more diverse downstream region is more appropriate for intraspecies comparisons.
The gene for actin as a highly conserved and functionally essential genetic element is developing... more The gene for actin as a highly conserved and functionally essential genetic element is developing into a major tool for phylogenetic analysis within a broad organismic range. We therefore propose a set of universally applicable primers that allow reliable amplification of actin genes. For primer construction the amino acid sequences of 57 actin genes comprising fungi, animals, plants and protists were analysed, aligned and used for the definition of six well-conserved regions which are suitable as priming sites in PCR amplification experiments. Ten primers were designed for specific in vitro amplification of actin gene fragments from a wide range of microorganisms. The corresponding gene fragments provide a strong basis to isolate nearly complete actin genes for further molecular characterization and for establishing phylogenies based on actin gene trees.
The randomly amplified polymorphic DNA technique (RAPD) is a modification of peR that uses short,... more The randomly amplified polymorphic DNA technique (RAPD) is a modification of peR that uses short, arbitrarily generated single primers to amplify genomic DNA. Amplified DNA-fragments are often polymorphic and can be used as individual, population-or species-specific markers. Because the RAPD technique requires a very high degree of reproducibility at the instrumentation level and with regard to buffer conditions, we propose to synthesize highly specific conventional PCR primers, the sequence of which is based on the primary diagnostic RAPD bands. In this communication we present a fast and convenient experimental strategy for converting the non-stringent RAPD conditions with their low annealing temperatures to stringent PCR conditions. Diagnostic RAPD bands were sequenced by a combination of chemical (Gilbert/ Maxam) and chain termination (Sanger) techniques. Based on this sequence information, highly specific oligonucleotide primers were synthesized. The value of this approach was demonstrated for the molecular diagnosis of the important rape seed (Brassica napus) pathogen Leptosphaeria maculans.
47 Polish isolates of the blackleg fungus Leptosphaeria maculans (Phoma lingam) were compared wit... more 47 Polish isolates of the blackleg fungus Leptosphaeria maculans (Phoma lingam) were compared with eight well-defined reference strains from Germany, France, Denmark, Australia and one Polish isolate of Phoma nigrificans. The isolates were tested (i) for growth characteristics, (ii) for their ability to form sirodesmins, (iii) for cellulolytic enzymes, and (iv) for pathotype-differentiating molecular markers generated by RAPD-PCR, PCR analysis with pathotype-specific primer pairs and PFGE.
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Papers by Kerstin Voigt
organism of mucormycosis, a rare but rapidly progressive infection in immunocompromised humans. The functionally annotated
genomic sequence consists of 74 scaffolds with a total number of 11,510 genes.
organism of mucormycosis, a rare but rapidly progressive infection in immunocompromised humans. The functionally annotated
genomic sequence consists of 74 scaffolds with a total number of 11,510 genes.