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Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    Akkermansia species : phylogeny, physiology and comparative genomics
    Ouwerkerk, J.P. - \ 2016
    Wageningen University. Promotor(en): Willem de Vos, co-promotor(en): Clara Belzer. - Wageningen : Wageningen University - ISBN 9789462577411 - 178
    akkermansia - akkermansia muciniphila - gastrointestinal microbiota - phylogeny - physiology - genomics - dna sequencing - nucleotide sequences - transcriptomes - antibiotic resistance - genome annotation - akkermansia - akkermansia muciniphila - microbiota van het spijsverteringskanaal - fylogenie - fysiologie - genomica - dna-sequencing - nucleotidenvolgordes - transcriptomen - antibioticaresistentie - genoomannotatie

    The gastrointestinal tract is lined with a mucus layer, which is colonized by a distinct mucosal microbial population. The anaerobic gut bacterium Akkermansia muciniphila is a well-described member of the mucosal microbiota and has been shown to be a human gut symbiont. In the mucus layer this gut symbiont is likely exposed to the oxygen that diffuses from mucosal epithelial cells. We showed that A. muciniphila has an active detoxification system to cope with reactive oxygen species and can use oxygen for respiration at nanomolar oxygen concentrations, with cytochrome bd as terminal oxidase.

    Until now, the type strain A. muciniphila MucT was the only cultured representative of this species. We isolated and characterized six new A. muciniphila strains from faecal samples of four different human subjects. These A. muciniphila strains showed minimal genomic and physiologic divergence while retaining their mucin degrading and utilisation capacities. Apart from the human gastrointestinal tract, we detected Akkermansia species in intestinal samples of numerous mammals. An additional ten new A. muciniphila strains were isolated from seven different mammalian species and showed high genomic and physiologic similarity to type strain A. muciniphila MucT. Apart from Akkermansia species, other Verrucomicrobia were identified within the gastrointestinal tract of non-human mammals. Furthermore, we obtained an Akkermansia isolate from the reticulated python, which had a similar mucin degrading capacity as the human strain A. muciniphila MucT but showed more efficient galactose utilization. On the basis of further phylogenetic, physiological, and genomic characterisations, strain PytT was found to represent a novel species within the genus Akkermansia, for which the name Akkermansia glycaniphilus sp. nov. is proposed.

    Overall, A. muciniphila strains isolated from intestinal samples of human and other mammals show very limited genomic and physiologic divergence. This together with the widely-spread global presence of A. muciniphila and the dependence on mucin for optimal growth, points towards a conserved symbiosis. This conserved symbiosis might be indicative for the beneficial role of this organism in respect to the host metabolic health. This is in line with the observation that A. muciniphila has been negatively associated with obesity and its associated metabolic disorders.

    In mice, treatment with viable A. muciniphila cells reversed high-fat diet-induced obesity. We described a scalable workflow for the preparation and preservation of high numbers of viable cells of A. muciniphila under strict anaerobic conditions for therapeutic interventions. Moreover, we developed various quality assessment and control procedures aimed to ensure the use of viable cells of A. muciniphila at any location in the world. These viable cells were used in a pilot study in humans in which no adverse events were observed. This is promising for future applications of A. muciniphila as a new therapeutic, leading towards the potential treatment of unhealthy states of the microbiota.

    Genetic diversity and evolution in Lactuca L. (Asteraceae) : from phylogeny to molecular breeding
    Wei, Z. - \ 2016
    Wageningen University. Promotor(en): Eric Schranz. - Wageningen : Wageningen University - ISBN 9789462576148 - 210
    lactuca sativa - leafy vegetables - phylogeny - genetic diversity - domestication - molecular breeding - genomes - dna - quantitative trait loci - evolution - lactuca sativa - bladgroenten - fylogenie - genetische diversiteit - domesticatie - moleculaire veredeling - genomen - dna - loci voor kwantitatief kenmerk - evolutie

    Cultivated lettuce (Lactuca sativa L.) is an important leafy vegetable worldwide. However, the phylogenetic relationships between domesticated lettuce and its wild relatives are still not clear. In this thesis, I focus on the phylogenetic relationships within Lactuca L., including an analysis of the wild Lactuca species that are endemic to Africa for the first time. The genetic variation of responses to salinity in a recombinant inbred line population, derived from a cross between the lettuce crop (L. sativa ‘Salinas’) and wild species (L. serriola), was investigated and the candidate gene in the identified QTL regions was further studied.

    In Chapter 1, I introduce and discuss topics related to genetic diversity and evolution in Lactuca, including an overview of lettuce cultivars and uses, its hypothesized domestication history, the taxonomic position of Lactuca, current status of molecular breeding in lettuce and mechanisms of salinity tolerance in plants, especially the High-affinity K+ Transporter (HKT) gene family.

    In Chapter 2, the most extensive molecular phylogenetic analysis of Lactuca was constructed based on two chloroplast genes (ndhF and trnL-F), including endemic African species for the first time. This taxon sampling covers nearly 40% of the total Lactuca species endemic to Africa and 34% of all Lactuca species. DNA sequences from all the subfamilies of Asteraceae in Genbank and those generated from Lactuca herbarium samples were used to elucidate the monophyly of Lactuca and the affiliation of Lactuca within Asteracaeae. Based on the subfamily tree, 33 ndhF sequences from 30 species and 79 trnL-F sequences from 48 species were selected to infer phylogenetic relationships within Lactuca using Randomized Axelerated Maximum Likelihood (RAxML) and Bayesian Inference (BI) analyses. In addition, biogeographical, chromosomal and morphological character states were analysed based on the Bayesian tree topology. The results showed that Lactuca contains two distinct phylogenetic clades - the crop clade and the Pterocypsela clade. Other North American, Asian and widespread species either form smaller clades or mix with the Melanoseris species in an unresolved polytomy. The newly sampled African endemic species probably should be excluded from Lactuca and treated as a new genus.

    In Chapter 3, twenty-seven wild Lactuca species and four outgroup species were sequenced using next generation sequencing (NGS) technology. The sampling covers 36% of total Lactuca species and all the important geographical groups in the genus. Thirty chloroplast genomes, including one complete (partial) large single copy region (LSC), one small single copy region (SSC), one inverted repeat (IR) region, and twenty-nine nuclear ribosomal DNA sequences (containing the internal transcribed spacer region ) were successfully assembled and analysed. A methodology paper for which I am co-author, but is not included in this thesis, of the sequencing pipeline was published: ‘Herbarium genomics: plastome sequence assembly from a range of herbarium specimens using an Iterative Organelle Genome Assembly (IOGA) pipeline’. These NGS data helped resolve deeper nodes in the phylogeny within Lactuca and resolved the polytomy from Chapter 2. The results showed that there are at least four main groups within Lactuca: the crop group, the Pterocypsela group, the North American group and the group containing widely-distributed species. I also confirmed that the endemic African species should be removed and treated as a new genus.

    In Chapter 4, quantitative trait loci (QTLs) related to salt-induced changes in Root System Architecture (RSA) and ion accumulation were determined using a recombinant inbred line population derived from a cross between cultivated lettuce and wild lettuce. I measured the components of RSA by replicated lettuce seedlings grown on vertical agar plates with different NaCl concentrations in a controlled growth chamber environment. I also quantified the concentration of sodium and potassium in replicates of greenhouse-grown plants watered with 100 mM NaCl. The results identified a total of fourteen QTLs using multi-trait linkage analysis, including three major QTLs associated with general root development (qRC9.1), root growth in salt stress condition (qRS2.1), and ion accumulation (qLS7.2).

    In Chapter 5, one of the identified QTL regions (qLS7.2) reported in Chapter 4 was found to contain a homolog of the HKT1 from Arabidopsis thaliana. I did a phylogenetic analysis of Lactuca HKT1-like protein sequences with other published HKT protein sequences and determined transmembrane and pore segments of lettuce HKT1;1 alleles, according to the model proposed for AtHKT1;1. Gene expression pattern and level of LsaHKT1;1 (L. sativa ‘Salinas’) and LseHKT1;1 (L. serriola) in root and shoot were investigated in plants growing hydroponically over a time-course. The measurements of Na+ and K+ contents were sampled at the same time as the samples used for gene expression test. In addition, I examined the 5’ promoter regions of the two genotypes. The results showed low expression levels of both HKT1;1 alleles in Lactuca root and relatively higher expression in shoot, probably due to the negative cis-regulatory elements of HKT1 alleles found in Lactuca promoter regions. Significant allelic differences were found in HKT1;1 expression in early stage (0-24 hours) shoots in and in late stage (2-6 days) roots. shoot HKT1;1 expression/root HKT1;1 expression was generally consistent with the ratios of Na+/K+ balance in the relevant tissues (shoot Na+/K+ divided by root Na+/K+).

    In Chapter 6, I summarize and discuss the results from previous chapters briefly. The implications of Chapter 2 and 3 for Lactuca phylogenetics are discussed, including some key characters for the diagnosis of species within Lactuca, the use of herbarium DNA for NGS technology, and perspectives into Lactuca phylogeny. Future perspectives of genome-wide association mapping for lettuce breeding were also discussed. Lastly, I propose to integrate phylogenetic approaches into investigations of allelic differences in lettuce, not just associated with salinity stress but also with other stressed and beneficial characters, both within and between species.

    Sexual development of Botrytis species
    Terhem, R.B. - \ 2015
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Jan van Kan. - Wageningen : Wageningen University - ISBN 9789462574144 - 188
    botrytis - plantenziekteverwekkende schimmels - geslachtsontwikkeling - fylogenie - genomica - transcriptomica - paarsystemen - schimmelmorfologie - nieuwe soorten - botrytis - plant pathogenic fungi - sexual development - phylogeny - genomics - transcriptomics - mating systems - fungal morphology - new species

    Sexual Development of Botrytis Species

    PhD Thesis

    Razak bin Terhem

    The fruiting bodies of species in the genus Botrytis are called apothecia. Apothecia are ascomas with an open cup shape on top of a stipe. Currently there is little information on processes occurring during apothecium development in Botrytis species. The aims of the research described in this thesis were to study the mechanisms involved in apothecium development of Botrytis cinerea, and to describe the morphology of Botrytis species and their fruiting bodies. Chapter 2 describes a genome-wide transcriptome analysis of different stages of apothecium development and a study on the function of MAT genes in apothecium development of B. cinerea. Functional analyses by targeted knockout mutagenesis revealed that the MAT1-1-1 gene and the MAT1-2-1 gene are both required for the initiation of sexual development. By contrast, mutants in the MAT1-1-5 gene and the MAT1-2-4 resulted in normal development of stipes which, however, were defective in the formation of an apothecial disk, asci and ascospores. Chapter 3 describes the functional analysis of three hydrophobin genes in sclerotium and apothecium development of B. cinerea. All three genes contribute to sclerotium and apothecium development. Chapter 4 describes the structure of the MAT1-1 and MAT1-2 locus in Botrytis elliptica and the morphology of apothecia of B. elliptica. Chapter 5 provides a morphological and phylogenetic description of Botrytis deweyae, the only species within the genus that behaves as an endophyte and in certain conditions is able to cause disease on Hemerocallis plants. Chapter 6 discusses the results presented in this thesis and puts them in a broader perspective. A model of processes and mechanisms involved in apothecium development is proposed.

    Restyling Alternaria
    Woudenberg, J.H.C. - \ 2015
    Wageningen University. Promotor(en): Pedro Crous; Pierre de Wit, co-promotor(en): J.Z. Groenewald. - Wageningen : Wageningen University - ISBN 9789462574106 - 250
    alternaria - taxonomie - fylogenie - moleculaire taxonomie - plantenziekteverwekkende schimmels - alternaria - taxonomy - phylogeny - molecular taxonomy - plant pathogenic fungi

    The omnipresent dematiaceous hyphomycete genus Alternaria is associated with a wide variety of substrates including seeds, plants, agricultural products, humans, soil and even the atmosphere. It includes saprophytic, endophytic and pathogenic species, among which multiple plant pathogens, post-harvest pathogens, and human pathogens (causative agents of phaeohyphomycosis and hypersensitivity reactions). Molecular studies reveal that the Alternaria complex comprises nine genera. Within this complex several genera are non-monophyletic and Alternaria species cluster into multiple distinct species clades, which are not always correlated with species-groups based on morphological characteristics. The most commonly reported species in literature and type species of the genus Alternaria, A. alternata, also comprises one such species-group. The small-spored Alternaria species within this group are mainly described based on morphology and / or host-specificity, but are difficult to distinguish based on molecular techniques alone. As A. alternata is considered as one of the most prolific producers of fungal allergens and is reported as pathogen on over 100 host plants, correct species identification is of utmost importance. The research presented in this thesis discusses the taxonomic status of Alternaria and its related genera, with a further focus on the two biggest and most important species complexes; the large-spored A. porri and small-spored A. alternata species complexes. With the phylogenies and classifications presented in this thesis, more robust and understandable taxonomy and nomenclature in Alternaria and allied genera within the Alternaria complex are created.

    Chapter 1 gives a general introduction to the genus Alternaria and related genera. The history of the genus and its economic importance as plant pathogen, post-harvest pathogen, causative agent of phaeohyphomycosis and common allergen causing hypersensitivity reactions are summarized. The introduction of the morphological species complexes, based on characters of the conidia, the pattern of chain formation, and the nature of the apical extensions of conidia are treated. Molecular studies recognise seven Alternaria species-groups within the Alternaria complex. Besides Alternaria, eight other genera are assigned to the Alternaria complex based on molecular and morphological studies.

    Chapter 2 focusses on the relationship of Alternaria and its closely related genera within the broader Alternaria complex. The phylogenetic lineages within the Alternaria complex are delineated based on nucleotide sequence data of parts of the 18S nrDNA (SSU), 28S nrDNA (LSU), the internal transcribed spacer regions 1 and 2 and intervening 5.8S nrDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase second largest subunit (RPB2) and translation elongation factor 1-alpha (TEF1) gene regions. The phylogenetic data reveal a Stemphylium clade sister to Embellisia annulata and a big Alternaria clade. The Alternaria clade contains six monotypic lineages and 24 internal clades, which are treated as sections of Alternaria. In order to create a stable phylogenetic taxonomy, and supported by i) a well-supported phylogenetic node in multiple analyses, ii) a high-similarity of clades within Alternaria based on SSU, LSU and ITS data, and iii) variation in the clade order between the different gene phylogenies, 13 genera are placed into synonymy with Alternaria. Embellisia annulata is synonymized with Dendryphiella salina, and together with D. arenariae placed in the new genus Paradendryphiella. The sexual genera Clathrospora and Comoclathris, with asexual forms linked to Alternaria, cluster within the Pleosporaceae, as does Alternaria, but outside Alternaria s. str. The genus Alternariaster, described to accommodate Alternaria helianthi, clusters within the Leptosphaeriaceae.

    Chapter 3 describes the reappraisal of the genus Alternariaster. Alternaria helianthi, the causal agent of leaf spot on Helianthus annuus (sunflower) was segregated from Alternaria based on conidial morphology, and placed in the new genus Alternariaster. A multi-gene phylogeny of parts of the ITS, LSU, RPB2 and GAPDH gene regions placed a fungal pathogen associated with leaf spot on Bidens sulphurea (yellow cosmos) in Brazil in close relation with Al. helianthi. Based on the close phylogenetic relation to Al. helianthi, but distinct morphological and pathogenicity characters, the fungal pathogen associated with leaf spot on B. sulphurea is newly described as Al. bidentis.

    Chapter 4 treats the Alternaria species which form the largest section of Alternaria, sect. Porri. This section contains almost all Alternaria species with medium to large conidia with long beaks, some of which are important plant pathogens. A multi-gene phylogeny on parts of the ITS, GAPDH, RPB2, TEF1 and Alternaria major allergen (Alt a 1) gene regions, supplemented with morphological and cultural studies, forms the basis for species recognition in this section. The polyphasic data reveal 63 species in sect. Porri, of which 10 are newly described, and 27 names are synonymized.

    Chapter 5 treats the small-spored Alternaria species, which reside in sect. Alternaria. A lot of confusion around the naming of species within this section exists, since the naming is mostly based on morphology and host-specificity, although the molecular variation is minimal. Whole genome sequencing, combined with transcriptome profiling and multi-gene sequencing of nine gene regions, SSU, LSU, ITS, GAPDH, RPB2, TEF1, Alt a 1, endopolygalacturonase (endoPG) and an anonymous gene region (OPA10-2), is used to create a clear and stable species classification in this section. The nine sequenced Alternaria genomes range in size from 32.0 - 39.1 Mb. The number of repetitive sequences varies significantly, with a relative low percentage of repeats within sect. Alternaria. The genome identity within sect. Alternaria is high, compared to the genome identity for isolates from other sections to the A. alternata reference genome. Similarly, a relative low percentage of single nucleotide polymorphisms (SNPs) were observed in genomic and transcriptomic sequences between isolates from sect. Alternaria, compared to the percentage of SNP’s found in isolates from different sections compared to the A. alternata reference genome. A set of core proteins was extracted from the genome and transcriptome data, and primers were designed on two eukaryotic orthologous group (KOG) protein loci with a relatively low degree of conservation within section Alternaria. The phylogenies from these two gene regions, KOG1058 and KOG1077, could not distinguish the described morphospecies within sect. Alternaria better than the phylogenies based on the nine commonly used gene regions for Alternaria. Based on genome and transcriptome comparisons and molecular phylogenies, Alternaria sect. Alternaria consists of only 11 phylogenetic species and one species complex. Thirty-five morphospecies are synonymized under A. alternata. The subclades that are formed by these isolates are incongruent between the different gene regions sequenced; no two genes show the same groupings for any of the over 100 isolates. A sequence-based identification guide is provided for the species which are now recognized in sect. Alternaria. None of the genes sequenced in this study can distinguish all of the species recognized here on its own.

    Chapter 6 investigates the molecular diversity of indoor Alternaria isolates in the USA, with the help of a phylogeographic / population genetic approach. Isolates collected throughout the USA were identified using ITS, GAPDH and endoPG gene sequencing, followed by genotyping and population genetic inference of the sect. Alternaria isolates and 37 reference isolates, using five microsatellite markers. Phylogenetic analyses revealed that 98 % (153 isolates) of the indoor isolates consisted of species from Alternaria sect. Alternaria. The remaining 2 % (three isolates) represented one sect. Infectoriae and two sect. Pseudoulocladium isolates. From the 153 isolates that belonged to sect. Alternaria, one could be assigned to A. burnsii, 15 to the A. arborescens species complex and the remaining 137 isolates were identified as A. alternata. Based on the microsatellite data, no specific indoor population could be distinguished. Population assignment analyses of the A. alternata isolates suggested that subpopulations did not exist within the sample, which we thus divided into four artificial subpopulations to represent four quadrants of the USA. Genotypic diversity was extremely high for all quadrants and a test for linkage disequilibrium suggested that A. alternata has a cryptic sexual cycle. The SouthWest-USA population displayed the highest level of uniqueness, based on private alleles. Intriguingly, the highest amount of gene flow, between SouthWest-USA and SouthEast-USA, correlated with the west-to-east movement of the antitrade winds. This suggests that indoor A. alternata isolates, although extremely diverse, have a continental distribution and high levels of gene flow over the continent.

    Chapter 7 discusses the data presented in this thesis. The implications of the performed studies are placed in a broader context, with a focus on the relation between morphology and the new species classification based on molecular tools and the use of genome data in contrast to multi-gene data.

    The butterfly plant arms-race escalated by gene and genome duplications
    Edger, P.P. ; Heidel-Fischer, H.M. ; Bekaert, K.M. ; Rota, J. ; Glockner, G. ; Platts, A.E. ; Heckel, D.G. ; Der, J.P. ; Wafula, E.K. ; Tang, M. ; Hofberger, J.A. ; Smithson, A. ; Hall, J.C. ; Blanchette, M. ; Bureau, T.E. ; Wright, S.I. ; dePamphilis, C.W. ; Schranz, M.E. ; Conant, G.C. ; Barker, M.S. ; Wahlberg, N. ; Vogel, H. ; Pires, J.C. ; Wheat, C.W. - \ 2015
    Proceedings of the National Academy of Sciences of the United States of America 112 (2015)27. - ISSN 0027-8424 - p. 8362 - 8366.
    evolutionaire genetica - co-evolutie - diversificatie - brassica - pieridae - papilionidae - glucosinolaten - fylogenie - evolutionary genetics - coevolution - diversification - brassica - pieridae - papilionidae - glucosinolates - phylogeny - diversity - defense - cytochrome-p450 - polymorphism - arabidopsis - metabolism - expression - speciation
    Coevolutionary interactions are thought to have spurred the evolution of key innovations and driven the diversification of much of life on Earth. However, the genetic and evolutionary basis of the innovations that facilitate such interactions remains poorly understood. We examined the coevolutionary interactions between plants (Brassicales) and butterflies (Pieridae), and uncovered evidence for an escalating evolutionary arms-race. Although gradual changes in trait complexity appear to have been facilitated by allelic turnover, key innovations are associated with gene and genome duplications. Furthermore, we show that the origins of both chemical defenses and of molecular counter adaptations were associated with shifts in diversification rates during the arms-race. These findings provide an important connection between the origins of biodiversity, coevolution, and the role of gene and genome duplications as a substrate for novel traits.
    Systematics, evolution and historical biogeography of the family Ochnaceae with emphasis on the genus Campylospermum
    Bissiengou, P. - \ 2014
    Wageningen University. Promotor(en): Marc Sosef, co-promotor(en): Lars Chatrou; L. Ngok Banak. - Wageningen : Wageningen University - ISBN 9789462572225 - 357
    ochnaceae - biosystematiek - taxonomie - evolutie - biogeografie - plantengeografie - fylogenie - taxonomische revisies - fylogenetica - ochnaceae - biosystematics - taxonomy - evolution - biogeography - phytogeography - phylogeny - taxonomic revisions - phylogenetics

    Abstract

    Ochnaceae s.l. is a family of trees, shrubs or rarely herbs widely distributed in tropical and subtropical forests and savannas of the Old and New World, and has about 500 species in 32 genera. The family is divided into three subfamilies: Medusagynoideae, Quiinoideae and Ochnoideae. We have provided, for the first time, a nearly complete molecular phylogenetic analysis of Ochnaceae s.l. resolving most of the phylogeny backbone of the family using five DNA regions. Based on this, dating analyses were performed using a secondary calibration, and relaxed molecular clock models. The historical biogeography of Ochnaceae s.l. was reconstructed using Dispersal-Vicariance Analysis and Bayesian Binary MCMC. The Neotropics were inferred as being the geographical origin of the family and the Old World was most likely colonized via the North Atlantic Land Bridge during a period when climatic conditions allowed establishment of a boreotropical flora. A full taxonomic revision of the continental African species of the genus Campylospermum has been prepared and additional historical biogeographic analyses were performed with a focus on the genus Campylospermum.

    Phylogeny and DNA-based identification in Phoma and related genera
    Aveskamp, M.M. - \ 2014
    Wageningen University. Promotor(en): Pedro Crous; Pierre de Wit. - Wageningen : Wageningen University - ISBN 9789461739148 - 206
    phoma - fylogenie - taxonomie - moleculaire taxonomie - identificatie - verklarende woordenlijsten - phoma - phylogeny - taxonomy - molecular taxonomy - identification - glossaries

    This thesis treats the taxonomy of a generic complex presently known as PhomaSacc. emend Boerema & Bollen. This group of fungi comprises more than 200 taxa at species or variety level that are characterised by the production of hyaline, non-septate conidial spores in pycnidial conidiomata. The genus is omnipresent in the environment, and exponents can be found on a wide range of host substrates.

    For many years the genus Phomawas the main research topic of a group of mycologists at the Dutch National Plant Protection Service. The studies conducted in the last decennia of the previous century culminated in a handbook that monographed the majority of the species in the above-mentioned generic complex. This handbook marked the end of the era in which the taxonomy of this genus mainly relied on morphological observations and cultural descriptions. However, it can also be regarded as the starting point of the present study. The aim of the present project was to integrate DNA-based identification methods into the taxonomic system established by the previously mentioned group of researchers. The major part of this study therefore deals with the validation of current generic and species concepts.

    An extensive literature review of the biology, taxonomy and identification methods to the species in this genus is provided in Chapter 2, with specific reference to the progress that has been made in Phomataxonomy after the publication of the abovementioned handbook. The advantages and disadvantages of the current taxonomical system are discussed. Furthermore, this chapter describes the general biology of the species in this fungal group, including their life cycles, distribution and host substrates. The importance of the genus for plant health and quarantine issues is illustrated, and the development of a rapid and robust identification technique based on DNA barcodes is advocated.

    Chapter 3treats species in Phomasection Peyronellaea. Species in this section are typified by the production of dictyochlamydospores, and thus have additional morphological characters to use in taxon delineation in comparison with species in the other Phomasections. All species in this group were subjected to a morphological re-examination and phylogenetic analyses employingITS, actin, and β-tubulin nucleotide sequences. Based on multi-gene analyses, Phomasection Peyronellaeacould not be maintained as a taxonomic entity, due to the polyphasic nature of taxa in this section. The morphological study revealed that for five species a taxonomic revision was required. A further five species appeared to be new to science, including Ph. microchlamydospora, Ph. omnivirens, and Ph. schachtii. Also the taxonomic noveltiesPh. coffeae-arabicaeand Ph. sancta are described here, and are allocated to the genus Peyronellaea, re-erected in Chapter 5.

    In Chapter 4the diversity among species and varieties belonging to the Ph. exiguaspecies complex is investigated. The Ph. exiguaspecies complex includes nine taxa at varietal level and four species that have a high morphological similarity both in vivoand in vitro, whilst historical relations with plant hosts cannot be maintained. Among this group, both omnipresent saprobes as well as host-specific plant pathogens are present – including the potato pathogen Ph. foveata.

    The diversity in this complex is studied by means of Internal Transcribed Spacer regions 1 & 2 and intervening 5.8S nrDNA (ITS) and actin nucleotidesequence analyses and a DNA fingerprinting technique rarely used to study fungal diversity. This technique, DNA Amplification Fingerprinting (DAF) employs short, arbitrary primers that form a loop, or a mini-hairpin, under specific temperature conditions and is frequently used in molecular plant breeding. The amplified DNA fragments were isolated and sequenced in order to develop taxon-specific markers and primer combinations based on the SCARs (Sequence Characterised Amplified Regions) and actin sequence data generated. These tools can aid rapid identification of this morphologically highly similar set of taxa.

    Two separate taxa were recognised within the type variety Ph. exiguavar. exigua. In the following chapter these taxa are described and all species and varieties in this complex are recombined into the new genus Boeremia.

    Chapter 5provides further details about the taxonomy and phylogeny of the species of interest, with a special focus on the taxa that are phylogenetically placed with Didymellaceae. In total 206 taxa were treated, of which 159 have affinities with Phoma.

    The genus is circumscribed in the first section of this chapter. The phylogeny was reconstructed using 28S nrDNA (Large Subunit) and 18S nrDNA (Small Subunit) sequence data. It was shown that the currently used Boeremaean subdivision of the phomoid taxa and the phylogeny were inconsistent, as the genus was highly polyphyletic. Species belonging to the form-genus Phomawere retrieved in as much as six distinct clades within Pleosporales. These clades even represent different families. The majority of the phomoid taxa, including the type species Ph. herbarumand most exponents of the sections Macrospora, Peyronellaea, Heterospora, and Phyllostictioides, were found in a single clade that represented the Didymellaceae. Most species that are associated with the Phomasections Plenodomusand Pilosacluster with the Leptosphaeriaceaeand Pleosporaceaeclades. Furthermore, some species were also found to cluster in the Sporormiaceaeand Cucurbitariaceaeclades.

    In the second part of this chapter, the phylogenetic variation of the species and varieties in Didymellaceaeis further assessed, using a phylogenetic reconstruction that is based on DNA sequences of the Large Subunit, ITS, and part of the β-tubulin (TUB) gene region. Besides the teleomorph genus Didymella, members of the teleomorph genera Leptosphaerulinaand Macroventuriawere also found to cluster in this clade. Based on the reconstructed phylogeny, Didymellaceaesegregate into at least 18 distinct clusters, of which many can be associated with specific morphological characters. Furthermore, a number of taxa did not match any of these clusters, suggesting that an evolutionary correct subdivision of the Phomaspecies in Didymellaceaeis even more complex. Taxa in four of these phylogenetic clusters were also defined well enough by means of morphology to elevate these groups to new or reinstalled genera, namely Stagonosporopsis, Epicoccum, Boeremiaand Peyronellaea. A total of 61 taxa were recombined and several new species of Phomawere introduced, namely Ph. brasiliensis, Ph. bulgarica, Ph. dactylidis, Ph. dimorpha, Ph. longicolla, Ph. minor, Ph. pedeiaeand Ph. saxea. Furthermore, two new varieties were described, Boeremia exigua var. gilvescensand B. exigua var. pseudolilacis.

    Finally, the results presented in this dissertation are highlighted and discussed inChapter 6. In total 13 species of Phomaand two taxa at varietal level were newly described during the course of this study. Moreover the taxonomic status of species in the form-genus Phomawere further clarified, and insight provided into the phylogenetic status of Didymellaceae, a fungal family that was recently established, comprising most species of Phoma, Ascochytaand Didymella. All macro- and micromorphological data obtained in this study, as well as the DNA sequences, were placed online in a publicly available polyphasic identification database (www.q-bank.eu). This will enable scientists and institutes involved in plant health to correctly identify phomoid species. Rapid identification of these species based on the tools and data generated in this study, can facilitate swift clearing of plant material and arable products during export and import, and prevent the spread of quarantine organisms.

    Phytopathogenic Dothideomycetes
    Crous, P.W. ; Verkley, G.J.M. ; Groenewald, J.Z. - \ 2013
    Utrecht, The Netherlands : CBS-KNAW Fungal Biodiversity Centre (Studies in mycology 75) - ISBN 9789070351960 - 406
    dothideomycetes - plantenziekteverwekkende schimmels - taxonomie - fylogenie - gastheerreeks - alternaria - cercospora - phoma - pseudocercospora - septoria - nieuwe soorten - nieuw geslacht - dothideomycetes - plant pathogenic fungi - taxonomy - phylogeny - host range - alternaria - cercospora - phoma - pseudocercospora - septoria - new species - new genus
    This volume of Studies in Mycology is dedicated to the plant health officers of the world, who are constantly confronted by a range of plant pathogenic fungi that cause devastating diseases of agricultural and forestry crops. Five main groups of fungi are dealt with, namely Alternaria, Cercospora, Phoma, Pseudocercospora and Septoria. In the first paper Phoma sections Plenodomus, Heterospora and Pilosa were reinvestigated, resulting in the introduction of several novel genera and species. The second paper deals with the paraphyletic genus Pseudocercospora; host specificity was considered for 146 species of Pseudocercospora occurring on 115 host genera from 33 countries. From these results we concluded that the application of European and American names to Asian taxa, and vice versa, was often not warranted. The third paper deals with the genus Cercospora, which contains more than 5 000 different species. Isolates used in the molecular phylogeny were obtained from 161 host species, 49 host families and 39 countries. Although some species were found to host-specific, others were isolated from a wide host range. The fourth paper deals with phylogenetic lineages within the genus Alternaria, which was revealed to represent a well-supported node containing 24 internal clades and six monotypic lineages. Several genera were placed in synonymy with Alternaria, for which 16 new sections were proposed. Two papers deal with the genus Septoria, which was shown to be poly- and paraphyletic, leading to the introduction of 15 new genera, and more than 40 new species. Although some species were shown to be highly specific, other taxa were revealed to occur on hosts in more than six different plant families. For all taxa investigated multi-gene DNA data were deposited in GenBank and other databases to expedite future identification of these plant pathogenic fungi. No single locus was found to be the ideal DNA barcode gene for these taxa, and species identification will have to be based on a combination of gene loci and morphological characters.
    The genus Gloriosa (Colchicaceae) : ethnobotany, phylogeny and taxonomy
    Maroyi, A. - \ 2012
    Wageningen University. Promotor(en): Jos van der Maesen, co-promotor(en): Lars Chatrou. - S.l. : s.n. - ISBN 9789461732446 - 190
    gloriosa - taxonomie - fylogenie - etnobotanie - zaden - plantensamenstelling - knollen - colchicine - economische botanie - gloriosa - taxonomy - phylogeny - ethnobotany - seeds - plant composition - tubers - colchicine - economic botany
    This thesis focuses on the ethnobotany, phylogeny and taxonomy of the genus Gloriosa L. over its distributional range. Some Gloriosa species are known to have economic and commercial value, but the genus is also well known for its complex alpha taxonomy. An appropriate taxonomy for this group is of great importance because it includes widely used species as traditional medicine, horticultural plants and sources of industrial and pharmaceutical chemical colchicine. The seeds and tubers of G. superba are valued as a commercial source of colchicine. The genus Gloriosa has considerable horticultural appeal because of the conspicuous inflorescence of its members and the ease with which taxa are propagated, introduced into new areas and hybridise in cultivation. G. carsonii, G. modesta, G. simplex and G. superba have been taken into cultivation as ornamental plants in several countries, including native countries of these species.
    Baculoviral and marsupial CPD photolyases: DNA repair proteins with a circadian clock function
    Biernat, M.A. - \ 2012
    Wageningen University. Promotor(en): Just Vlak; G.T.J. van der Horst, co-promotor(en): Monique van Oers; I. Chaves. - S.l. : s.n. - ISBN 9789461732163 - 113
    baculoviridae - lepidoptera - buideldieren - lyasen - dna-herstel - flavoproteinen - circadiaan ritme - fylogenie - genetische analyse - evolutie - baculoviridae - lepidoptera - marsupials - lyases - dna repair - flavoproteins - circadian rhythm - phylogeny - genetic analysis - evolution

    Baculoviruses infect insects and are highly virulent, host specific and environmentally safe, and therefore, are used as biocontrol agents of pest insects. Their effective use in the field is hampered, however, by the ultraviolet (UV) light, which induces cyclobutane pyrimidine dimers (CPDs) in (viral) DNA. CPD photolyases are enzymes that repair CPDs with the help of visible light in a process called photoreactivation. The baculovirus Chrysodeixis chalcites nucleoplyhedrovirus (ChchNPV) possesses two photolyase genes, Cc-phr1 and Cc-phr2. Only Cc-phr2 encodes an enzymatically active photolyase. CPD photolyases are members of the cryptochrome/photolyase family (CPF), which consists of two types of proteins that are structurally conserved, but have different functions. Whereas photolyases repair DNA, cryptochromes work as photoreceptors or regulators of the circadian clock. In mammals, cryptochromes act as inhibitors of CLOCK/BMAL1-driven transcription, and hence, regulate proper functioning of the 24h oscillator. This thesis focuses on the origin and function of baculovirus CPD photolyases. Phylogenetic analysis indicated that a single horizontal gene transfer from an ancestral lepidopteran host led to the current presence of phr genes in a particular clade of the family Baculoviridae. A next study examined whether ChchNPV occlusion bodies (OBs) profited from the photolyases in becoming less UV sensitive. However, this was not the case, as the OBs’s DNA could not be photoreactivated after UV irradiation. This led to the hypothesis that Cc-PHRs may have another function in baculovirus pathogenesis, or alternatively, may induce behavioral changes in the host. Considering the homology to cryptochromes, a possible role of CPD photolyases in the molecular oscillator (‘clock’) was studied. This revealed that Cc-PHR2 and the Potorous tridactylus photolyase (PtCPD-PL), in contrast to Cc-PHR1 and Arabidopsis thaliana (6-4) photolyase, were able to substitute for mammalian cryptochromes. Both Cc-PHR2 and PtCPD-PL inhibited CLOCK/BMAL1-driven transcription and dampened the oscillation of cultured fibroblasts, probably due to the observed interaction with the CLOCK protein. Moreover, both CPD photolyases revived oscillations in arrhythmic cryptochrome knockout (Cry1-/-/Cry2-/-) mouse dermal fibroblasts and livers, showing that Cc-PHR2 and PtCPD-PL can work as true cryptochromes. Chromatin- and co-immunoprecipitation experiments showed that Cc-PHR2 and PtCPD-PL do not prevent CLOCK to bind chromatin nor do they disrupt CLOCK/BMAL1 heterodimer formation. Therefore, these proteins probably use a similar mechanism as mammalian cryptochromes to drive the clock and their overall structure rather than their amino acid sequence and/or domain availability determines their functionality. The gathered data advanced our understanding of the functional evolution of the CPF and showed that the studied class II CPD photolyases are dually functional proteins that repair DNA, but can also regulate the circadian clock in a similar manner as cryptochromes.

    Key words: photolyases, cryptochromes, circadian clock, DNA repair, functional evolution, baculoviruses

    The genus Phytophthora; phylogeny, speciation and host specificity
    Kroon, L.P.N.M. - \ 2010
    Wageningen University. Promotor(en): Francine Govers; Pierre de Wit, co-promotor(en): W.G. Flier. - [S.l. : S.n. - ISBN 9789085856689 - 184
    phytophthora - plantenziekteverwekkers - fylogenie - soortvorming - gastheerspecificiteit - plantenziekten - plantenziekteverwekkende schimmels - phytophthora infestans - phytophthora - plant pathogens - phylogeny - speciation - host specificity - plant diseases - plant pathogenic fungi - phytophthora infestans
    Pormotie-onderzoek naar de fylogenie, soortsvorming en waardplantspecificiteit in het geslacht Phytophthora.
    Highlights of the Didymellaceae: a polyphasic approach to characterise Phoma and related pleosporalean genera
    Aveskamp, M.M. ; Gruyter, H. de; Woudenberg, J. ; Verkley, G. ; Crous, P.W. - \ 2010
    Utrecht : CBS Fungal Biodiversity Centre (Studies in mycology 65) - ISBN 9789070351793 - 65
    dothideales - phoma - taxonomie - fylogenie - identificatie - pleosporaceae - coelomycetes - dothideales - phoma - taxonomy - phylogeny - identification - pleosporaceae - coelomycetes
    Genomic support for speciation and specificity of baculoviruses
    Jakubowska, A.K. - \ 2010
    Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Monique van Oers. - [S.l. : S.n. - ISBN 9789085856207 - 136
    baculovirus - baculoviridae - insecten - soortvorming - fylogenie - gastheerspecificiteit - genexpressieanalyse - baculovirus - baculoviridae - insects - speciation - phylogeny - host specificity - genomics
    Keywords: baculovirus, insects, speciation, genomics, phylogeny, host specificity

    The Baculoviridae comprise a large family of double-stranded DNA viruses infecting
    arthropods. In this thesis two baculoviruses, Leucoma salicis nucleopolyhedrovirus
    (LesaNPV) and Agrotis segetum (Agse) NPV, were characterized in detail. Both viruses are
    potential biocontrol agents of the insects from which they were isolated. A close genetic
    relationship between LesaNPV and Orgyia pseudotsugata multiple NPV (OpMNPV) was
    found. O. pseudotsugata is known from North America and contains two baculoviruses,
    OpSNPV and OpMNPV. L. salicis is a European insect species that was accidentally
    introduced in the beginning of the 20th century into North America. Results from the current
    study suggest that LesaNPV was imported along with L. salicis into North America, where it
    infected O. pseudotsugata and adapted to this new host in coexistence with OpSNPV. As
    such, this case provides a snapshot of baculovirus evolution through speciation. The genome
    sequence of AgseNPV showed a striking co-linearity with Spodoptera exigua (Se) MNPV,
    although these viruses vary in biological properties such as host specificity. AgseNPV can
    infect S. exigua orally, but SeMNPV is not infectious for A. segetum larvae. SeMNPV causes
    a systemic infection in A.segetum only when the midgut barrier is bypassed through injection
    of the virus into the hemocoel. SeMNPV was able to enter A. segetum midgut epithelial cells
    and to transcribe its early genes, but was unable to replicate and produce progeny virus in
    these cells. The AgseNPV / SeMNPV case provides an excellent model to study baculovirus
    specificity by analyzing the changes in the genome sequence that lead to the differences in
    host range. The collected data support the view that molecular characterization is essential for
    proper virus classification and for assessing the phylogenetic relationships with other viruses.
    A phylogenetic re-evaluation of Dothideomycetes
    Schoch, C.L. ; Spatafora, J.W. ; Lumbsch, H.T. ; Huhndorf, S.M. ; Hyde, K.D. ; Groenewald, J.Z. ; Crous, P.W. - \ 2009
    Utrecht, the Netherlands : CBS-KNAW (Studies in mycology 64) - ISBN 9789070351786 - 220
    pezizomycotina - fylogenetica - fylogenie - evolutie - taxonomie - moleculaire taxonomie - pezizomycotina - phylogenetics - phylogeny - evolution - taxonomy - molecular taxonomy
    This volume presents a re-evaluation of phylogenetic relationships within the class Dothideomycetes, which is by far the largest and arguably most phylogenetically diverse class within the largest fungal phylum, Ascomycota.
    On the natural and laboratory evolution of an antibiotic resistance gene
    Salverda, M.L.M. - \ 2008
    Wageningen University. Promotor(en): Rolf Hoekstra, co-promotor(en): Arjan de Visser; John van der Oost. - [S.l.] : S.n. - ISBN 9789085049999 - 144
    evolutie - bèta-lactamase - plasmiden - recombinatie - bacteriën - fenotypen - mutagenese - fylogenie - moleculaire genetica - selectie - antibioticaresistentie - evolution - beta-lactamase - plasmids - recombination - bacteria - phenotypes - mutagenesis - phylogeny - molecular genetics - selection - antibiotic resistance
    TEM-1 ß-lactamase is one of the most notorious antibiotic resistance enzymes around. It exists at high frequencies in antibiotic-resistant bacteria around the world and confers resistance to ß-lactam antibiotics, including penicillins (e.g. ampicillin) and cephalosporins. The enzyme displays a remarkable phenotypic plasticity in response to the introduction of new drugs; within a few years after the clinical debut of most new ß-lactam antibiotics resistance conferring variants of TEM-1 are isolated. Such a shift in resistance phenotype is typically caused by just a few amino acid substitutions. Until today, more than 150 variants of TEM-1 with a unique amino acid sequence have been identified.
    Because of the clear link between genotype and phenotype (i.e. level of resistance or fitness) and because of the ease of selecting for increased antibiotic resistance, TEM-1 has been used as a model in studies that seek new methods to optimize proteins. These studies combine the power of in vitro mutagenesis and in vivo selection and have resulted in a wealth of information about which mutations can increase resistance when the enzyme is exposed to an antibiotic that it initially hydrolyzes inefficiently. At a later stage, these techniques were adopted and used to repeat and predict the natural evolution of TEM-1 under various selective conditions. Recently, TEM-1 is increasingly being used as an experimental model for the study of fundamental evolutionary questions, particularly those that benefit from the direct relationship between genotype and phenotype.
    In this thesis, both the natural and laboratory evolution of TEM-1 are studied. The aim of the laboratory work is to increase our understanding of the way in which adaptive mutations interact. For this purpose, TEM-1 is mutagenized using error-prone PCR, which creates variation in the resulting copies of the TEM-1 gene. Mutated gene-copies are placed in bacteria which are subsequently selected for increased resistance to cefotaxime (an antibiotic that TEM-1 hydrolyzes poorly). By repeating this process multiple times in independent experiments, the mutations and mutational trajectories involved in the increase of cefotaxime resistance are studied. At a fundamental level, this has lead to a better understanding of the nature of mutation interaction and its consequences for evolutionary contingency and constraint. Evidence indicating that certain ‘silent’ mutations (i.e. mutations that alter the codon sequence but not the amino acid that the respective codon encodes) can also play a role in increased resistance was found in these data as well.
    A phylogenetic study of the sequences of the ~150 different TEM-alleles that have been isolated in hospitals and clinics so far indicates that recombination has played a significant role in the evolution of TEM-alleles, contrary to what is often assumed. Furthermore, amino acid substitutions present in these clinical isolates are compared to those found in laboratory evolution studies of TEM-1, in order to investigate to what extent laboratory evolution can be used as a predictive tool for the natural evolution of antibiotic resistance genes. This overview indicates that laboratory evolution very accurately repeats the natural evolution of TEM-1. Based on these findings, predictions are made about substitutions that may appear in future clinical TEM-isolates, and directions are given how laboratory evolution can be exploited as a predictive tool most efficiently.
    Phylogeny, detection, and mating behaviour of Mycosphaerella spp. occurring on banana
    Arzanlou, M. - \ 2008
    Wageningen University. Promotor(en): Pedro Crous; Pierre de Wit, co-promotor(en): L.H. Zwiers; Gert Kema. - [S.l. : S.n. - ISBN 9789085048008 - 171
    musa - plantenziekteverwekkende schimmels - mycosphaerella - ramichloridium - fylogenie - detectie - paringsgedrag - heterothallisme - morfotaxonomie - moleculaire diagnostiek - musa - plant pathogenic fungi - mycosphaerella - ramichloridium - phylogeny - detection - mating behaviour - heterothallism - morphotaxonomy - molecular diagnostics
    The genus Mycosphaerella is phylogenetically heterogeneous, and has been linked to more than 30 anamorphic genera. Plant pathogenic species of Mycosphaerella are among the most common and destructive plant pathogens, causing considerable economic losses on a wide range of hosts by invading leaf and stem tissue, and distorting the host plant physiology. The Sigatoka leaf spot disease complex of bananas involves three related ascomycetous fungi: Mycosphaerella fijiensis, M. musicola and M. eumusae. Besides these three primary agents of the Sigatoka disease complex, several additional species of Mycosphaerella (or their anamorphs) have been reported from Musa. This thesis provides insight into various taxonomic aspects of the genus Mycosphaerella in general and Mycosphaerella species pathogenic on banana in particular, with an emphasis on biodiversity, phylogeny and evolutionary plasticity of mating type loci.
    Chapter 1 gives an introduction to the genus Mycosphaerella, with specific reference to the Sigatoka disease complex of banana, and further provides a brief review on sexual reproduction in ascomycetous fungi and Mycosphaerella in particular. Chapter 2 describes the biodiversity and phylogenetic relationships among the Mycosphaerella species constituting the Sigatoka disease complex of banana. From the data presented in this chapter, it is clear that the Sigatoka disease complex is caused by a multitude of Mycosphaerella species. More than 20 species of Mycosphaerella or associated anamorphs were identified on banana. Eight out of these twenty species were described for the first time, and five species were previously described to occur on other host crops. Chapter 3 describes the development and use of molecular tools to detect and quantify the major constituents of the Sigatoka disease complex (M. fijiensis, M. musicola
    and M. eumusae) in planta.
    In Chapter 4 I clarified the taxonomic position of Ramichloridium musae, the casual agent of tropical speckle disease of banana, and morphologically similar genera such as Periconiella, Veronaea and Rhinocladiella to Mycosphaerella. The phylogeny inferred from 28S nrDNA sequence data revealed the genus Ramichloridium to be heterogeneous. The phylogenetic analyses place Ramichloridium species in five different orders within the Ascomycota, with the type species residing in the Capnodiales. Furthermore, Periconiella was shown to reside in Mycosphaerella; Rhinocladiella and Veronaea in the Chaetothyriales.
    In Chapter 5 I characterised the mating type loci of the primary agents of the Sigatoka disease complex of banana by the application of a chromosome-walking strategy, genomic analyses and bioinformatics. The data revealed that the mating type loci of these three heterothallic Mycosphaerella species are characterised by an expansion in size and the presence of a number of new genes which are presumably unique to members of Mycosphaerella. These analyses were extended to study the evolutionary history of the mating type loci in M. fijiensis, M. musicola and M. eumusae.
    Finally, the results of this thesis are discussed with a broader outlook in Chapter 7. Various taxonomic aspects of the genus Mycosphaerella in general and Mycosphaerella species pathogenic on banana in particular are addressed, with emphasis on biodiversity and phylogeny. Furthermore, the evolutionary history of sexual reproduction of the three major Mycosphaerella species on banana is discussed. Altogether, the data presented in this thesis suggest a two step evolutionary history for the causal agents of the Sigatoka disease complex of banana.
    An integrative algorithmic approach towards knowledge discovery by bioinformatics
    Alako Tadontsop, F.B. - \ 2008
    Wageningen University. Promotor(en): Jack Leunissen. - [S.l. : S.n. - ISBN 9789085048190 - 124
    bio-informatica - nomenclatuur - computeranalyse - nucleotidenvolgordes - algoritmen - fylogenetica - moleculaire biologie - fylogenie - classificatie - genexpressieanalyse - datamining - microarrays - ontologieën - bioinformatics - nomenclature - computer analysis - nucleotide sequences - algorithms - phylogenetics - molecular biology - phylogeny - classification - genomics - data mining - microarrays - ontologies
    In this thesis we describe different approaches aiding in the utilization of the exponentially growing amount of information available in the life sciences. Briefly, we address two issues in molecular biology, on sequence analysis, and on text mining. The former issue addresses the problem how to determine remote sequence homology especially when the sequence similarity is very low. For this a visualisation tool is introduced that combines sequence alignment, domain prediction and phylogeny. The second topic on text mining centres on the question how to unambiguously formulate queries for efficient information retrieval. It tackles the problem of gene nomenclature — one in two gene symbols being ambiguous - by introducing a new text-clustering- and taxonomy-based disambiguation methodology.
    Phylogenetic relationships within the phylum Nematoda as revealed by ribosomal DNA, and their biological implications
    Holterman, M.H.M. - \ 2007
    Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Hans Helder. - [S.l.] : S.n. - ISBN 9789085048800 - 208
    nematoda - ribosomaal dna - fylogenetica - klassering volgens erfelijke eigenschappen - fylogenie - plantenparasitaire nematoden - vrijlevende nematoden - dorylaimidae - chromadoridae - tylenchidae - zeenematoden - single nucleotide polymorphism - nematoda - ribosomal dna - phylogenetics - cladistics - phylogeny - plant parasitic nematodes - free living nematodes - dorylaimidae - chromadoridae - tylenchidae - marine nematodes - single nucleotide polymorphism
    Nematodes – “eel worms”; members of the phylum Nematoda – can be considered as a success story within the Metazoa (multicellular, heterotrophic eukaryotes in which cells lack cell walls): they are speciose and – probably - the most numerous group of multicellular animals on our planet. Nematodes are present in virtually all terrestrial, freshwater and marine habitats. Nematodes are trophically diverse; they may feed on bacteria, fungi/oomycetes, algae and protozoa, other nematodes or on a combination of these (omnivores), or live as facultative or obligatory parasites of plants or animals. As they are abundant, ubiquitous and occupy several trophic levels, they play an important role in the soil food web. Nematode parasites of animals affect billions of humans and livestock, while plant parasites such as cyst, root knot and lesion nematodes cause large agricultural losses worldwide.
    Despite their undisputed ecological and economical relevance, the systematics of the phylum Nematoda is far from established. One of the aims of this research was to further elucidate nematode phylogeny using molecular data. First a phylogenetic tree was constructed of 349 taxa, spanning the entire phylum Nematoda, on the basis of full length small subunit ribosomal DNA (SSU rDNA) sequences. A series of mostly well-supported bifurcations defined twelve major clades, whereas the most basal clade was defined by representatives of the Enoplida and Triplonchida. Our analysis confirmed the paraphyly of the Adenophorea. Furthermore it was found that the SSU rDNA from representatives of the distal clades evolved at a higher rate than the SSU rDNA from the basal clades. In the meantime, a substantial number of sequences was added to our overall SSU rDNA nematode alignment - both public data (GenBank) and data generated by ourselves (≈ 1,500 sequences in total; February 2008). It is noted that the clade division as proposed in 2006 on the basis of “only” 349 taxa still seems to be valid.
    Subsequent research focused on three specific groups; Dorylaimia, Chromadoria and Tylenchomorpha. Within the suborder Dorylaimina, the SSU rDNA provided an exceptionally low phylogenetic signal, and - therefore – a part (≈ 1,000 bp) of the more variable large subunit ribosomal DNA (LSU rDNA) was analyzed. In most cases nematode relationships could be elucidated with good support, although some areas in the trees remained unresolved. Generally speaking the results of molecular phylogenetics corresponded fairly well with classical nematode taxonomy. The main exception was the order Dorylaimida where twelve subclades could be distinguished which bore little resemblance to classical taxonomy. Furthermore the suitability of ribosomal DNA for a (semi-) quantative molecular identification method was demonstrated using quantitative PCR (q-PCR) and primers designed to specifically amplify members of the order Mononchida and the potato cyst nematodes Globodera pallida and G. rostochiensis.
    Plant parasitism has arisen several times within the phylum Nematoda (once in the Triplonchida, at least three times in the Dorylaimida and at least twice in the Tylenchomorpha). The long-standing and generally accepted hypothesis states that plant parasites evolved from fungal feeding ancestors. However, while in most cases plant parasites were associated with fungal feeding nematodes, this hypothesis could neither be confirmed nor denied with the results of our phylogenetic analyses. In the case of two Dorylaimida (Pungentus and Longidorella), however, the ancestor was probably an omnivore. The analysis of this problem was substantially hampered by the lack of knowledge on feeding behavior of basal Tylenchomorpha.
    Presumably, the common ancestor of the nematodes lived in a marine environment and - if this assumption is correct - the transition to a limnoterrestrial environment must have taken place at least once. Surprisingly, analysis of the Chromadoria (minus the Rhabditida) revealed that transitions from a thalassic to a limnoterrestrial habitat (and vice versa) have taken place at least 11 times in the Chromadoria. Given their frequency these transitions are apparently fairly easy to achieve for nematodes and the possible adaptations involved were discussed.
    Nematodes vary widely in their responses to environmental disturbance, making them good bio-indicators of soil health. Yet it is not known with certainty which traits are responsible for tolerance to stress in nematodes. A framework was laid out to study correlations between nematode traits and stress tolerance. Furthermore the importance of accounting for the confounding effects of phylogeny was demonstrated. This is a first step towards a transparent, ecological grouping of free-living nematodes.
    It is worthwhile mentioning that - on the basis of the rDNA-based molecular framework described in this PhD thesis - DNA sequences signatures were identified for nearly all North-West European terrestrial and freshwater nematodes families. The relationship between quantitative PCR signal and numbers of individuals has been established for nearly all families and a first testing of DNA barcode-based community analysis is planned for spring 2008.

    Fylogenetische SSU rDNA-analyse van het fylum Nematoda
    Holterman, M.H.M. ; Elsen, S.J.J. van den; Megen, H.H.B. van; Wurff, A.W.G. van der; Helder, J. - \ 2007
    Gewasbescherming 38 (2007)4. - ISSN 0166-6495 - p. 150 - 154.
    nematoda - ongewervelde dieren - fylogenie - evolutie - moleculaire taxonomie - parasitisme - trichodoridae - plantenparasitaire nematoden - globodera - soorten - identificatie - nematoda - invertebrates - phylogeny - evolution - molecular taxonomy - parasitism - trichodoridae - plant parasitic nematodes - globodera - species - identification
    Nematoden vormen één van de meestgevarieerde en succesvolle diergroepen ter wereld. Ze zijn waarschijnlijk de meest talrijke dieren op aarde, komen in uiteenlopende milieus voor (zowel terrestrische als marien) en spelen een belangrijke rol in het ecosysteem. De verscheidenheid van voedingstypes en habitats maken deze groep ook erg interessant vanuit een evolutionair oogpunt. Ons onderzoek richt zich op het uitzoeken van de evolutionaire verwantschappen tussen nematoden aan de hand van het ribosomaal DNA - een neutraal gen dat niets te maken heeft met dier- of plantparasitisme - en de evolutie van kenmerken als voedingstypen, stresstrolerantie en de overgang van een marien leefmilieu naar het land. Nematodentaxonomie is een onderzoeksveld dat sinds het begin in beweging is geweest. De geconserveerde morfologie en de vaak moeilijk waarneembare kenmerken bemoeilijken de reconstructie van de evolutie van de nematoden. Dit heeft tot gevolg gehad dat de nematodensystemetiek steeds veranderde en er bijna net zoveel classificaties als taxonomen zijn. De laatste jaren is er veel veranderd door de opkomst van de moleculaire fylogenie. Het gebruik van DNA-sequenties - in het geval van vaak het small subunit ribosomal DNA (SSU rDNA)-gen - om de evolutie te traceren heeft geleid tot nieuwe inzichten en een hernieuwde interesses in nematodenevolutie
    Botrytis species on flower bulb crops: phylogeny, genetic variation and host specificity
    Staats, M. - \ 2007
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Jan van Kan. - [S.l.] : S.n. - ISBN 9789085045687 - 167
    bloembollen - botrytis - plantenziekteverwekkende schimmels - fylogenie - genetische variatie - gastheerspecificiteit - botrytis tulipae - genetische merkers - ornamental bulbs - botrytis - botrytis tulipae - plant pathogenic fungi - phylogeny - genetic markers - genetic variation - host specificity

    Fungi of the genus Botrytis (teleomorph Botryotinia) can cause serious damage in a large range of ornamental crops. Except for B. cinerea, Botrytis species that are pathogenic on flower bulb crops are host-specific, i.e. each species is able to infect only one or a few closely related host species. This thesis mainly focuses on the economically important species B. elliptica and B. tulipae, the causal agents of 1eaf blight in lily and tulip, respectively.

    Molecular markers were developed that allowed for the unambiguous identification of the Botrytis species of interest and to study their intraspecific variation (Chapter 2, 3 & 4). Chapter two describes a classification of the genus Botrytis based on DNA sequence data of three protein-coding genes {RPB2, GSPDB and HSP60). The phylogenetic analysis that encompassed all 22 species of the genus corroborated the classical species delineation. In addition, the hybrid status of B. allii (B. byssoidea X B. aclada) was confirmed. A comparison of Botrytis and angiosperm phylogenies showed that cospeciation of pathogens and their hosts have not occurred. Rather, it is proposed that host shifts have occurred during Botrytis speciation.

    Chapter three describes three molecular typing methods to differentiate isolates of B. tulipae, B. elliptica and B. cinerea. Restriction analysis of the intergenic spacer (IGS) region was the least discriminatory, followed by DNA sequencing of five gene regions (G3PDH, RPB2, HSP60, H3 and EF-Ia) and the internal transcribed spacer (ITS) region. Amplified fragment length polymorphism (AFLP) analysis appeared the most discriminatory method. It was, therefore, used to assess the genotypic diversity among field isolates of B. elliptica and B. tulipae from the Netherlands (Chapter 4). In addition, analyses were performed to infer their modes of reproduction. Isolates of both species were sampled during successive growth seasons from experimental field plots in Lisse and from several other locations in the Netherlands. The genotypic diversity for B. elliptica was high and clonal genotypes were found only within growing seasons. Linkage disequilibrium analyses strongly supported the occurrence of genetic recombination in the field, most likely as a result of sexual reproduction. Multilocus association analyses, however, provided no evidence for random mating.

    B. tulipae has a mainly clonal population structure, as evidenced by the low genotypic diversity, the repeated recovery of clonal genotypes over long distances and over different years, and the strong multilocus associations. However, the level of linkage disequilibrium is lower than expected for a strictly clonal organism. Therefore, it is possib]e that some level of recombination would have resulted in generating new genotypes.

    In chapters 5 and 6 the roles of Nepl-like proteins {NLPs) as determinants of host specificity are investigated. Two NLF-encoding genes, designated NEPl and NEP2, were present in all Botrytis species (Chapter 5). Both NLPs may have different functions as their sequence similarity is low and because they contain different types of posttranslational modification motifs. While both NLPs are moderately conserved and are overall under purifying selection, a number of amino acid residues were predicted to be under positive selection based on inferences using maximum likelihood models. The functional effects of these amino acid replacements on the NEP protein activity remain to be resolved.

    Chapter 6 describes the functional characterization of NLP genes from B, elliptica. Mutants of B. elliptica in which BeNEPl or BeNEP2 was replaced showed normal virulence on lily leaves. In addition, the B. elliptica NLPs that were produced in the yeast Pichia pastoris were not toxic to monocots, including lily. These results show that NLPs are not essential virulence factors and do not function as host-selective toxins for B. elliptica.

    Chapter 7 presents a general discussion of the thesis and provides future directions to study the evolution, life cycles and pathogenicity of Botrytis species on flower bulb crops.

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