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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.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    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.
    The Ascomycota tree of life: a phylum-wide phylogeny clarifies the origin and evolution of fundamental reproductive and ecological traits
    Schoch, C.L. ; Sung, G.H. ; López-Giráldez, F. ; Townsend, J.P. ; Miadlikowska, J. ; Hofstetter, V. ; Robbertse, B. ; Brandon Matheny, P. ; Kauff, F. ; Wang, Z. ; Gueidan, C. ; Andrie, R.M. ; Trippe, K. ; Ciufetti, L.M. ; Wynns, A. ; Fraker, E. ; Hodkinson, B.P. ; Bonito, G. ; Groenewald, J.Z. ; Arzanlou, M. ; Hoog, G.S. de; Crous, P.W. ; Hewitt, D. ; Pfister, D.H. ; Peterson, K. ; Gryzenhout, M. ; Wingfield, M.J. ; Aptroot, A. ; Suh, S.O. ; Blackwell, M. ; Hillis, D.M. ; Griffith, G.W. ; Castlebury, L.A. ; Rossman, A.Y. ; Lumbsch, H.T. ; Lücking, R. ; Büdel, B. ; Rauhut, A. ; Diederich, P. ; Ertz, D. ; Geiser, D.M. ; Hosaka, K. ; Inderbitzin, P. ; Kohlmeyer, J. ; Volkmann-Kohlmeyer, B. ; Mostert, L. ; O'Donnell, K. ; Sipman, H. ; Rogers, J.D. ; Shoemaker, R.A. ; Sugiyama, J. ; Summerbell, R.C. ; Untereiner, W. ; Johnston, P.R. ; Stenroos, S. ; Zuccaro, A. ; Dyer, P.S. ; Crittenden, P.D. ; Cole, M.S. ; Hansen, K. ; Trappe, J.M. ; Yahr, R. ; Lutzoni, F. ; Spatafora, J.W. - \ 2009
    Systematic Biology 58 (2009)2. - ISSN 1063-5157 - p. 224 - 239.
    rna-polymerase-ii - multiple sequence alignment - fungal lineages - cleistothecial fungi - classification - subunit - pezizomycotina - divergence - morphology - eukaryotes
    We present a 6-gene, 420-species maximum-likelihood phylogeny of Ascomycota, the largest phylum of Fungi. This analysis is the most taxonomically complete to date with species sampled from all 15 currently circumscribed classes. A number of superclass-level nodes that have previously evaded resolution and were unnamed in classifications of the Fungi are resolved for the first time. Based on the 6-gene phylogeny we conducted a phylogenetic informativeness analysis of all 6 genes and a series of ancestral character state reconstructions that focused on morphology of sporocarps, ascus dehiscence, and evolution of nutritional modes and ecologies. A gene-by-gene assessment of phylogenetic informativeness yielded higher levels of informativeness for protein genes (RPB1, RPB2, and TEF1) as compared with the ribosomal genes, which have been the standard bearer in fungal systematics. Our reconstruction of sporocarp characters is consistent with 2 origins for multicellular sexual reproductive structures in Ascomycota, once in the common ancestor of Pezizomycotina and once in the common ancestor of Neolectomycetes. This first report of dual origins of ascomycete sporocarps highlights the complicated nature of assessing homology of morphological traits across Fungi. Furthermore, ancestral reconstruction supports an open sporocarp with an exposed hymenium (apothecium) as the primitive morphology for Pezizomycotina with multiple derivations of the partially (perithecia) or completely enclosed (cleistothecia) sporocarps. Ascus dehiscence is most informative at the class level within Pezizomycotina with most superclass nodes reconstructed equivocally. Character-state reconstructions support a terrestrial, saprobic ecology as ancestral. In contrast to previous studies, these analyses support multiple origins of lichenization events with the loss of lichenization as less frequent and limited to terminal, closely related species.
    Comparative analysis of genetic incompatibility in Aspergillus niger and Podospora anserina
    Pal, K. - \ 2007
    Wageningen University. Promotor(en): Rolf Hoekstra, co-promotor(en): Fons Debets. - [S.l.] : S.n. - ISBN 9789085046554 - 135
    aspergillus niger - deuteromycotina - pezizomycotina - incompatibiliteit - genetische analyse - aspergillus niger - deuteromycotina - pezizomycotina - incompatibility - genetic analysis
    Reproduction is of crucial importance for living organisms, from bacteria to humans. Except for mammals and birds, both sexual and asexual reproduction is known almost at every level of thephylogenetictree. A special case of asexual reproduction in filamentous fungi is the so calledparasexualcycle, whenhyphaeof different individuals fuse and the genetic material of different origin can be mixed. The black mouldAspergillusniger is a versatile organism used in industry and sometimes found as opportunistic pathogen. This is an asexual species, and most of the natural isolates are unable to undergo theparasexualcycle, in other words strains are (vegetatively) incompatible. Unlike the A.niger thePodosporaanserina is a strictly sexual species and used as a model organism for studying features ofparasexualcycle. In this study the genetic background of vegetative incompatibility and remnants of presumed ancient sexual stage of A.niger and features of vegetative incompatibility in P.anserina were investigated and compared.
    Basisboek paddenstoelen
    Dam, N.J. ; Kuyper, T.W. ; Dam, M. - \ 2006
    Utrecht : KNNV - ISBN 9789050112413 - 150
    paddestoelen - basidiomycotina - pezizomycotina - identificatie - determinatietabellen - nederland - schimmels - basidiomycotina - pezizomycotina - fungi - mushrooms - identification - keys - netherlands
    Phylogeny and taxonomy of Phaeoacremonium and its relatives
    Mostert, L. - \ 2006
    Wageningen University. Promotor(en): Pedro Crous, co-promotor(en): E.C.A. Abeln. - [S.l.] : S.n. - ISBN 9789085044604 - 249
    diaporthales - pezizomycotina - deuteromycotina - fylogenie - taxonomie - phaeohyphomycose - humane ziekten - medische mycologie - determinatietabellen - plantenziekteverwekkende schimmels - diaporthales - pezizomycotina - deuteromycotina - phylogeny - taxonomy - keys - phaeohyphomycosis - human diseases - medical mycology - plant pathogenic fungi
    Species of Phaeoacremonium are known vascular plant pathogens causing wilt and dieback of woody hosts.  The most prominent diseases in which they are involved are Petri disease and esca, which occur on grapevines and are caused by a complex of fungi, including Phaeomoniella chlamydospora and several species of Phaeoacremonium . Various Phaeoacremonium species are also opportunistic on humans, where they are known to cause phaeohyphomycosis. 

    The genus Togninia was confirmed as the teleomorph of Phaeoacremonium by means of morphology, sexual compatibility, and DNA phylogeny. Subsequently, the genus Togninia was monographed along with its Phaeoacremonium anamorphs. Ten species of Togninia and 22 species of Phaeoacremonium were treated. Several new species of Togninia were found during the course of the study, namely T. argentinensis , T. austroafricana , T. krajdenii , T.parasitica , T.rubrigena and T. viticola . New species of Phaeoacremonium includePm. alvesii, Pm. amstelodamense,Pm. argentinense ,Pm. australiense,Pm. austroafricanum, Pm. griseorubrum,Pm.krajdenii,Pm. novae-zealandiae , Pm . iranianum ,Pm. scolyti, Pm. sphinctrophorum,Pm.subulatum,Pm. tardicrescens,Pm . theobromatisandPm. venezuelense . Species were identified based on their cultural and morphological characters, supported by DNA data derived from partial sequences of the actin and β-tubulin genes. Phylogenies of the SSU and LSU rRNA genes were used to determine whether Togninia has more affinity with the Calosphaeriales or the Diaporthales . These results confirmed that Togninia had a higher affinity to the Diaporthales than the Calosphaeriales . Examination of type specimens revealed that T.cornicola , T.vasculosa , T . rhododendri, T . minima var. timidula and T.villosa , were not members of Togninia . New combinations such as Calosphaeria cornicola,Calosphaeriarhododendri , Calosphaeriatransversa , Calosphaeriatumidula,Calosphaeriavasculosa and Jattaeavillosawere therefore proposed to accommodate these species.

    The correct and rapid identification of Phaeoacremonium species is important tofacilitate the understanding of their involvementin plant as well as human disease. A rapid identification method was developed for the 22 species of Phaeoacremonium currently recognised. It involved the use of 23 species-specific primers, including 20 primers targeting the β-tubulin gene and three targeting the actin gene. These primers can be used in 14 multiplex reactions. Additionally a multiple-entry electronic key based on morphological, cultural andb-tubulin sequence data was developed to facilitate routine species identification. This database can be accessed online athttp://www.cbs.knaw.nl/phaeoacremonium.htm. With this identification tool phenotypic and sequence data can be used to identify the different Phaeoacremonium species. Separate dichotomous keys were provided for the identification of the Togninia and Phaeoacremonium species based on phenotypic characters. Additionally, keys for the identification of Phaeoacremonium -like fungi and the genera related to Togninia were also provided. 

    The mating strategy of several Togninia species was investigated with ascospores obtained from fertile perithecia produced in vitro . Togninia argentinensis and T.novae-zealandiae have homothallic mating systems, whereas T. austroafricana , T. krajdenii , T. minima , T.parasitica , T. rubrigena and T. viticola are heterothallic. The species predominantly isolated from diseased grapevines are Pm. aleophilum , Pm. parasiticum and Pm. viticola . Perithecia of two of these species, T. minima and T. viticola , have been found on grapevines in the field, indicating that these species recombine in vineyards. 

    The genetic diversity among isolates ofPa.chlamydospora on grapevines was investigated by means of Amplified Fragment Length Polymorphisms (AFLPs). Phaeomoniellachlamydospora isolates were collected from different positions on the same vine, different vines within a vineyard and different vineyards withinSouth Africa. Selected isolates from various grape producing countries, namelyAustralia,France,Italy,Iran,New Zealand,SloveniaandU.S.A.were also included. The overall low level of genetic variation confirmed asexual reproduction to be dominantin the field. Different genotypes were found among isolates ofPa.chlamydospora within the same grapevine, suggesting multiple infections from different inoculum sources. Isolates from different countries had a high percentage of similarity and clustered together, indicating the absence of genotype-geographic structure. The presence of the same genotype in different vineyards and production areas suggests that long-range dispersal through aerial inoculum or infected plant material played an important role in genotype distribution. 

    Seven species of Togninia and 15 species of Phaeoacremonium were newly described during the course of this study. The data obtained in this study, as well as the newly developed dichotomous and online polyphasic keys will enable scientists to correctly identify all the known species and also provide a reference database to which new species can be added in future. The newly reported host ranges and distributions, together with pathogenicity data, will enable scientists to identify species of possible quarantine concern.

    On the ecology and evolution of fungal senescence
    Maas, M.F.P.M. - \ 2005
    Wageningen University. Promotor(en): Rolf Hoekstra, co-promotor(en): Fons Debets. - [S.l.] : S.n. - ISBN 9789085042846 - 118
    pezizomycotina - neurospora - veroudering - verouderen - ecologie - evolutie - mitochondria - plasmiden - celbiologie - mutaties - genetica - pezizomycotina - neurospora - senescence - aging - ecology - evolution - mitochondria - plasmids - cellular biology - mutations - genetics
    Aging evolves in the shadow of natural selection: Since the efficiency of natural selection declines with age, organisms will over the course of generations accumulate intrinsic, genetic factors that have a negative effect only late in life. This is generally known as the 'mutation accumulation' theory of aging. Should these factors additionally have apleiotropic, positive effect early on in life, for example on fertility, they could even befavoredby natural selection. This is known as the 'antagonisticpleiotropy' theory of aging. Aging is thus expected to be a multi-causal process resulting from intrinsic factors with negative effects late in life and possibly additional, positive effects early in life. It can be seen as the result of a lack of investment in somatic maintenance, a legacy of an organism's evolutionary past.

    In contrast to unitary organisms like most animals, modular organisms like plants, fungi and colonial invertebrates should not be subject to aging: In these organisms, there is no clear distinction between germ line and soma. Because the germ line should be immortal, in modular organisms aging or senescence is generally not expected, though parts or modules may be subject to aging or senescence. Though this is rare, there are striking examples oforganismalsenescence in fungi and plants, in which all parts of an individual die at the same time.

    This thesis deals with aging in two genera of filamentous fungi:NeurosporaandPodospora. It deals with the question whether there are similarities, both at the proximate or mechanistic level and at the ultimate or evolutionary level, between aging processes in fungi and aging processes as we know them from animals. It is shown that, at least in the pseudo-homothallic filamentousascomycetePodosporaanserina, aging is an intrinsic andmulticausalprocess as may be expected. An analysis of natural variation in life span shows that the main source of variation in life span corresponds to the presence or absence of mitochondrial plasmids, molecular parasites that interfere with respiration. Variation that arises spontaneously in the laboratory often corresponds to mitochondrial mutations in the electron transport chain. The latter mutations are all associated with the induction of alternative; nuclear encoded respiratory pathways and this leads via a yet unknown route to a stabilization of the otherwise unstable mitochondrial genome, a reduced level of reactive oxygen species as well as a reduced energy level. These mutations hence confer longevity at the cost of fertility. In addition to spontaneous mutations and chemical modifications of the electron transport chain, a dietary  reduction in the amount of glucose can extend life span in fungi. The latter effect is strongly reduced by the presence of a type of mitochondrial plasmid that interferes with respiration, which indicates that it is strongly dependent on properly functioning mitochondria. The latter underlines the critical role of mitochondria in the fungal senescence
    Genomic conflicts in Podospora anserina = Genomische conflicten in Podospora anserina
    Gaag, M. van der - \ 2005
    Wageningen University. Promotor(en): Rolf Hoekstra, co-promotor(en): Fons Debets. - s.l. : S.n. - ISBN 9789085042556 - 152
    pezizomycotina - schimmels - genomen - genetica - plasmiden - meiotic drive - meiose - uitkruisen - pezizomycotina - fungi - genomes - genetics - plasmids - meiotic drive - meiosis - outcrossing
    This thesis deals with genomic conflicts raised by selfish elements in the ascomycete fungus Podospora anserina .Genomic conflicts arise when the effects of the selfish elements are opposite to the interests of the other parts of the genome. Two types of selfish elements are studied as well as certain characteristics of Podospora involved in the population dynamics of these elements, such as vegetative and sexual incompatibility, senescence and outcrossing.The natural habitat of Podospora anserina is dung of herbivores where it has an optimum growth temperature of 27 °C. The fungus can only reproduce sexually and the ascospores are the products of meiosis as well as the next generation of the fungus. Perithecia or fruiting bodies contain asci with four linearly arranged ascospores, which provide unique opportunities to analyse abnormal segregation and makes this fungus one of the genetic model organisms. Most ascospores are capable of completing the lifecycle of the fungus, as they contain two nuclei, each with one of the two mating types. This fungal trait is called pseudo or secondairy homothallism, and it allows sexual offspring to be produced by either selfing or outcrossing. Sometimes smaller single mating type ascospores are formed containing one nucleus and less cytoplasmic content and mitochondria. Colonies from these spores must outcross with another isolate to produce offspring. The fungal isolates used in this thesis were sampled from dung around Wageningen, theNetherlandsduring 1990-1997, but also some older French isolates dating from 1937 were used.

    The first selfish elements studied in this thesis are linear plasmids with homology to pAL2-1, a 8.3 kb plasmid previously found in this fungus. Linear plasmids are parasitic autonomous replicating genetic elements. In filamentous fungi they reside in the mitochondria. Most plasmids are cryptic or have a negative fitness effect on the host. However the pAL2 plasmid found in P. anserina has been associated with a longevity phenotype, though recently also negative effects were found. Homologous linear plasmids and plasmid families were present in 21 percent of the population and detected over several years. Half of the plasmid containing isolates possesseda single plasmid and the other half a plasmid family consisting of multiple plasmid copies. The lifespan of the isolates was generally uncorrelated with the presence of the plasmid. One isolate showed an increased lifespan, but noinserted plasmid sequences were detected in the mitochondrial DNA, as was the case for the longevity inducing pAL2-1 plasmid. We have looked at the dynamics of plasmid transmission for these plasmids.Vertical transfer of the plasmids to the ascospores occurs only via the maternal line. This transfer is inefficient as up to 40% lose all plasmids in an outcrossing situation and up to 20% retain only the basic plasmid family member in presence of a plasmid family. No difference in efficiency of plasmid transfer to the dikaryotic and the smaller monokaryotic ascospores was detected. Plasmid transmission was also foundindependent of the genetic background of the strains.Loss of plasmids via the sexual route is compensated by horizontal transmission of the plasmid through hyphal contact between different isolates. Horizontal transfer occurs efficiently in both vegetative compatible and incompatible situations. Vegetative incompatibility can be macroscopically observed as a 'barrage', a contact zone of lysed hyphal cells. Vegetative incompatibility is thought to have evolved as a mechanism to hamper spread of mobile selfish elements and parasites. Our experiments show that vegetative incompatibility is not a perfect barrier against this type of selfish elements.

    The other selfish elements studied in this thesis are meiotic drive factors or segregation distorters. Segregation distorters are transmitted into the progeny in excess of the fair Mendelian proportion of 50 %, by actively destructing the alternative allele. Genomic conflicts arise by hitchhiking of genes with deleterious fitness effects. Meiotic drive in Podospora ischaracterized by the abortion of two of the four spores in the ascus.Seven different groups of meiotic drive elements or Spore killer types were identified and characterized. Among 99 isolates from nature, six of these meiotic drive elements occurred in our local population. All drive elements comprise 23% of the natural population of P. anserina in Wageningen, The Netherlands and most elements can be retrieved over the years. Spore-killer type Psk-7 was also present in a French strain dating from 1937 and exists for more than 60 years. No resistance to meiotic drive was observed and all other isolates found so far are sensitive to spore killing. Each type of Spore killer differs in the percentage of asci that show killing, ranging from 50 to 95% two-spored asci, and in their mutual interactions. The aborted ascospores quickly degrade after spore wall formation, except for the Psk-3 group where they remain visible as tiny shriveled ascospores, indicating different abortion factors or timing for killing. The Psk-3 group also shows a variable percentage of two-spored asci within each perithecium. Spore-killer interactions show either mutual resistance ( i.e. no abortion is found if an allele of either Spore killer is present) or dominant epistasis ( i.e. one killer acts as a sensitive type). Genetic mapping could assign most Spore-killer types to linkage group III where they are not tightly linked to the centromere.

    Several possible models that explain the spore killing mechanism in Podospora anserina were examined. Repeatedbackcrossing of Spore killers to the same sensitive isolate produces strains with the same genetic background. Spore killers that belong to the same killer type or show mutual resistance become vegetative compatible to each other during backcrosses. On the other hand, Spore killers that show dominant epistasis, as well as the sensitive strain remained vegetative incompatible. This suggests a common mechanism for spore killing, possibly related to vegetative incompatibility, although the precise genetic nature of the correlation is not yet clear.The Podospora genome was screened for homologues of genes known to be involved in silencing in fungi. Genes were found for all silencing mechanisms (RIP, MSUD, Quelling) known in the related fungus Neurospora . However, the possible role of silencing by methylation of genes during the killing process was excluded by experiments using the drug 5-azacytidine, which both removes methylation and prevents de-novo methylation. No effect of the 5-azacytidine treatments was found on spore-killing frequency for all Spore-killer types. Also the consequences of formation of dikaryotic ascospores for expression of spore killing were examined. Crosses were used of Spore killers with a marker that increases the number of monokaryotic spores to up to eight. Spore-killer types Psk-2 , Psk-3 (Wa27) and Psk-4 produced some asci containing more than four spores in these crosses. In such asci sensitive nuclei were able to survive and resist the meiotic drive system, indicating incomplete penetrance of the spore-killing mechanism. Spore killing in the other killer types ( Psk-1, Psk-5, Psk-6 and Psk-7 ) were full penetrant; only asci with four or less spores could be detected. Here the killing mechanism works similar on all asci. Furthermore the effect of low temperatures (22 ºC) on spore killing was tested in this Chapter. Psk-2 dramatically decreased the percentage of killing at this temperature to almost zero. Other Spore-killer types were not affected by temperature. Based on all characteristics and interactions of the Spore killers we propose that spore killing in Podospora may be an example of post-segregational killing due to Toxin-Antitoxin mechanisms. A Spore Killer produces both a persisting stable toxin and a less stable antitoxin. In ascospores where the Spore Killer is absent the antitoxin disappears more quickly than the toxin, leading to abortion of the spores. Variable killing percentages can be explained by the strict balance between toxin and antitoxin and the timing of shutdown of the genes involved.

    Meiotic drive is only possible in an outcrossing situation. Podospora is in principle capable of both outcrossing and selfing. However to what extent the fungus outcrosses in nature is unknown. The likelihood of outcrossing was assessed for the secondary homothallic ascomycete Podospora anserina. We examined the extent of vegetative en sexual compatibility between wild type strains. The number of vegetative compatibility groups (VCG's) in the population was estimated based on the incompatibility reactions between isolates in our survey using accumulation curve extrapolation and the non-parametric Chao1 formula. The estimated number of VCG's compared to the maximum number of VCG's based on the currently known vegetative incompatibility genes suggest regular outcrossing in this fungus. Also the difference in sexual compatibility reactions of mating types of the same isolate assumes outcrossing takes place. Options for outcrossing in P. anserina were experimentally verified . Both single mating type monokaryotic and dikaryotic double mating type mycelial cultures proved capable of outcrossing, showing no preference for either genotype. This indicates that fertilization by selfing and outcrossing uses the same pathway. Outcrossing percentages between 1-5 percent were found in unmanipulated natural situations of ascospores on dung. The number of monokaryotic ascospores found in Spore-killer ( Psk ) strains was significantly higher than in other isolates, showing an enhancement to outcrossing in these strains.

    The findings in this thesis contribute to the understanding of the population dynamics and evolution of two types of selfish elements, linear plasmids and segregation distorters, in the ascomycete fungus Podospora anserina. Furthermore it increases the knowledge on genomic conflicts caused by these types of selfish elements in general
    Biological control of Botrytis spp. by Ulocladium atrum through competitive colonisation of necrotic plant tissues
    Köhl, J. - \ 2004
    Wageningen : Plant Research International - 232
    botrytis - pezizomycotina - biologische bestrijding - epidemiologie - schimmelantagonisten - botrytis - pezizomycotina - biological control - epidemiology - fungal antagonists
    Zorgvuldig omgaan met resistentie tegen appelschurft
    Schouten, H.J. - \ 2003
    De Fruitteelt 93 (2003)18. - ISSN 0016-2302 - p. 10 - 11.
    appels - plantenziekteverwekkende schimmels - schurft (bij dieren) - gewasbescherming - ziekteresistentie - epidemiologie - pezizomycotina - steriliseren - venturia inaequalis - apples - plant pathogenic fungi - plant protection - disease resistance - epidemiology - sterilizing - ?
    Appelrassen met schurftresistentie lopen kans deze resistentie te verliezen als verkeerd met deze resistentie wordt omgesprongen. Belangrijke rubrieken in dit artikel zijn: 1) De levenscyclus van schurft op appel; 2) Virulente schurftstammen; 3) Biologische factoren die het proces van onwerkzaam worden van Vf-resistentie vertragen; 4) Ascospoorvorming en geslachtelijke voortplanting; 5) Ontsmetten van Vf-rassen
    Meiotic sister chromatid cohesion and recombination in two filamentous fungi
    Heemst, D. van - \ 2000
    Agricultural University. Promotor(en): C. Heyting; H.W.J. van den Broek. - S.l. : S.n. - ISBN 9789058083180 - 127
    moleculaire genetica - meiose - chromatiden - zusterchromatidenuitwisseling - recombinatie - schimmels - emericella nidulans - pezizomycotina - mutanten - dna-sequencing - molecular genetics - meiosis - chromatids - sister chromatid exchange - recombination - fungi - emericella nidulans - pezizomycotina - mutants - dna sequencing

    Homologous recombination and sister chromatid cohesion play important roles in the maintenance of genome integrity and the fidelity of chromosome segregation in mitosis and meiosis. Within the living cell, the integrity of the DNA is threatened by various factors that cause DNA-lesions, of which DNA double-strand breaks (DSBs) are considered particularly deleterious. The causative agents can be of endogenous origin, such as metabolically produced free radicals, and of exogenous origin, such as ultraviolet light and ionizing radiation. The accurate repair of DSBs is important to prevent chromosomal fragmentation, translocations and deletions. Of the sophisticated (networks of) DNA repair pathways that have evolved, homologous recombination, which repairs the DSB by copying information from an intact homologous DNA-template, is considered one of the most accurate. In mitotic G2, the sister chromatid is preferentially used as a template for recombinational repair of DSBs.

    DSBs can also arise as normal intermediates in several DNA repair and recombination pathways, including meiotic recombination. In meiotic recombination (in yeast), the cells actively induce large numbers of DSBs, and channel their search for a homologous template towards a non-sister chromatid of the homologous chromosome. In each pair of homologous chromosomes, at least one DSB is repaired by reciprocal exchange of precisely corresponding segments of non-sister chromatids (crossing over), whereas additional DSBs in the same chromosome pair are repaired by either reciprocal or non-reciprocal exchange. The reciprocal exchanges between non-sister chromatids (visible as chiasmata) are essential for the proper disjunction of homologous chromosomes during the first meiotic division (meiosis I).

    When I started my investigations for this thesis, it was recognized that reciprocal exchanges as such could not direct the proper segregation of homologous chromosomes at meiosis I; some "glue" should keep the chiasmata in place, either by binding to the chiasmata, or by maintaining cohesion between the sister chromatids distal to the chiasmata. Although mutants existed that appeared to be defective in the production of this glue, its nature remained unknown. In this thesis, I have tried to identify components involved in meiotic sister chromatid cohesion and recombination, to analyze the interplay between these two processes in meiosis and to gain insight into their relationship with mitotic DNA repair and recombination.

    In chapter 1 , I explain the choice of the experimental model systems that I used for the research described in this thesis. All investigations were performed in two filamentous fungi, namely Sordaria macrospora and Aspergillus nidulans . The most important reason for the choice of these two fungi was that mutants were available (or easily obtainable) that were defective in meiotic sister chromatid cohesion and/or recombination, and that it should be feasible to clone the corresponding wild-type genes by means of transformation complementation of the mutant defects. S. macrospora had the additional advantage of a well-developed cytology and A. nidulans had the additional advantages of well-developed molecular genetic tools and the presence of a parasexual cycle in addition to the sexual cycle. This latter feature offers the possibility of analyzing mitotic allelic recombination. Moreover, A. nidulans is one of the two known organism that do not assemble synaptonemal complexes (SCs) during meiotic prophase and that do not display positive crossover interference. The choice for both S. macrospora and A. nidulans would thus make it possible to compare the role(s) of genes involved in meiotic sister chromatid cohesion and/or recombination in a organism with and one without SCs.

    In chapter 2 , we describe the cloning of the SPO76 gene of S. macrospora by transformation complementation of the meiotic defects of the spo76-1 (non-null) mutant. It was known that this mutant displayed defects in meiotic sister chromatid cohesion, meiotic recombination and mitotic DNA-repair. Furthermore, we analyzed the localization of the Spo76 protein throughout wild-type mitosis and meiosis and performed a detailed analysis of the spo76-1 mutant phenotype. We show that Spo76p is chromosome-associated during all stages of mitosis and meiosis, except at metaphase(s) and anaphase(s). During mitosis, Spo76p disappears from the chromosomes at prometaphase. During meiotic prophase I, Spo76p is more abundant than during any other cell cycle stage, and localizes preferentially close to the chromosome axes. Spo76p disappears from the chromosomes at diplotene. In the spo76-1 mutant, we observed a transient defect in chromosome organization at (mitotic) prometaphase: the duration of this stage was prolonged and chromosome morphology was abnormal. Strikingly, chromosomal regions with defects in both sister chromatid cohesion and chromosome compaction alternated with regions with apparently normal cohesion and compaction. We speculate that the mitotic prophase to metaphase transition involves forces that tend to disrupt cohesion and that Spo76p promotes the maintenance of a minimum of cohesion in combination with chromosome compaction. Likewise, we observed in meiotic prophase of spo76-1 , from late leptotene on, that sister chromatid cohesion and chromosome compaction were coordinately affected on a regional basis. Regions with widely split axial elements alternated with regions with unsplit segments of axial elements. These unsplit segments could form stretches of SC, which contained rare late recombination nodules (late RNs: ultrastructurally recognizable enzyme-complexes involved in the later steps of meiotic recombination). Whereas the number of late RNs was strongly reduced in spo76-1 , early RNs (as recognized by immunocytochemical labelling of Rad51 and Dmc1) occurred at only slightly reduced levels in the mutant and persisted longer. This suggest that spo76-1 is deficient in some intermediate step of meiotic recombination. The role of Spo76p in the meiotic leptotene/zygotene transition may be related to its role during the mitotic prophase/metaphase transition, in that the leptotene/zygotene transition might also bring along forces that tend to disrupt cohesion, and that Spo76p is also needed for maintenance of cohesion during this meiotic transition. Spo76p may have an additional role in late meiotic prophase because in spo76-1 , meiotic sister chromatids separated completely from diplotene on, whereas in wild type this does not occur before anaphase II.

    The predicted protein encoded by the SPO76 gene is evolutionary conserved from fungi to man; the highest percentage of amino acid identity (44%) was found with the BIMD protein of A. nidulans. The A. nidulansbimD6 mutant was previously identified as a conditional lethal mutant with a (lethal) mitotic chromosome segregation defect at high temperature and a (non-lethal) DNA repair deficiency phenotype at low temperature.

    In chapter 3 , we demonstrate by heterologous complementation that the SPO76 gene of S. macrospora can complement both the temperature and the MMS (methyl methane sulphonate) sensitivities of bimD6 in A. nidulans , implying direct functional homology between the mitotic roles of two proteins. We also show that, like spo76-1 , bimD6 mutants do not form sexual spores (ascospores). However, bimD6 mutants display, unlike spo76-1 , disturbances in premeiotic development and form only few asci. In the few asci that entered meiosis, sister chromatids separate prematurely, but the extent of meiotic sister chromatid separation is less severe in bimD6 than in spo76-1 . In addition, whereas the mitotic localization of the two proteins was roughly similar, the meiotic localization of the two proteins showed some important differences. Unlike Spo76p in S. macrospora , BIMD in A. nidulans was not more abundant during meiotic prophase than in the mitotic cycle, and did not localize preferentially close to the chromosome axes during the pairing of homologous chromosomes. Moreover, SPO76 could not complement the sexual sporulation defects of bimD6 in A. nidulans, and vice versa , BIMD could not restore the sexual sporulation defects of the S. macrospora spo76-1 mutant. These results indicate that Spo76p and BIMD may differ in a species-specific manner with respect to their meiotic roles. The species-specific aspects of the roles of Spo76p and BIMD6 in meiosis are possibly related to the differences in meiotic chromosome organization between the two fungi (see above; chapter 1 ): in contrast to S. macrospora , A. nidulans does not form SCs and does not display positive interference of meiotic crossovers.

    We also show that bimD6 mutants are hypersensitive to X-rays in addition to their elevated sensitivities to UV and MMS, but only when dividing cells are exposed to these agents. The bimD6 mutant thus closely resembles recombination-deficient mutants of A. nidulans , such as uvsC114 (the uvsC gene of A. nidulans is homologous to RAD51 of Saccharomyces cerevisiae ; chapter 4 ). We have therefore compared bimD6 with uvsC114 regarding defects in mitotic recombination. When assayed for allelic recombination, bimD6 and uvsC114 yielded similar results. In both mutants, the absolute frequencies of allelic recombination were strongly reduced, although the distribution of recombinants among the various classes was comparable to wild type. However, when assayed for intrachromosomal conversions, bimD6 and uvsC114 produced different results. Intrachromosomal conversions between interrupted duplications were significantly reduced in uvsC114 , but occurred at wild-type frequencies in bimD6 . We propose that BIMD is required for homologous recombination when the template is located on another (sister or non-sister) chromatid, but not when the template is available in close proximity on the same sister chromatid or the same chromatin domain/loop. The repair machinery may thus be obliged to cooperate with cohesion complexes (which are probably located at the borders between chromatin domains/loops) if the homologous template lies outside an intrachromatid loop domain. In contrast, uvsC was required for both types of repair.

    In chapter 4 , we describe the cloning of the uvsC gene of A. nidulans by transformation complementation of the mitotic repair defects of a uvsC114 mutant. Furthermore, we disrupted the entire uvsC gene, and we compared the phenotypic effects of the resulting null mutation with those of uvsC114 . The predicted UVSC protein shows 67% amino acid identity with Rad51p of S. cerevisiae and 27% amino acid identity with the RecA protein of Escherichia coli. These proteins are involved in strand invasion and exchange during homologous recombination. We found that in the absence of DNA-damaging agents, the uvsC gene was transcribed at a higher level in the uvsC114 mutant than in wild-type. Transcription of uvsC was inducible by MMS in wild-type and uvsC114 mutant strains. We compared the mitotic and meiotic phenotypes of mutants carrying the uvsC114 point mutation (a deletion of 6 bp in core domain I) with those of the uvsC null mutant. The uvsC null mutant was more sensitive to UV and MMS than uvsC114 , indicating that uvsC114 is not a null mutation. The sexual developmental phenotypes of the two mutants also differed. In the uvsC null mutant, sexual development was disturbed before the onset of meiosis, whereas in uvsC114 it was blocked in meiotic prophase. We observed large, multi-nucleated cells in older cleistothecia of the uvsC null mutant These cells possibly represent degenerated croziers, which might have been blocked at premeiotic S-phase. In S. cerevisiae , disruption of RAD51 has no effect on mitotic growth and causes arrest at meiotic prophase I, whereas in mouse, disruption of Rad51 results in embryonic lethality. In A. nidulans , disruption of uvsC had no effect on mitotic growth, and caused an arrest at a premeiotic stage of sexual development. Disruption of RAD51 -homologous genes thus has different effects in different organisms.

    In chapter 5 , we describe the isolation and characterization of sexual sporulation mutants of A. nidulans . Vegetative spores were treated with a high dose of UV (1.5 % survival) and surviving colonies were plated on supplemented minimal medium. Colonies that did not display aberrant vegetative growth were visually screened for the appearance of "barren" fruiting bodies (=fruiting bodies without or with only few ascosores). This screen (1250 colonies analyzed) yielded 20 mutants with the desired phenotype. After two successive rounds of backcrosses with wildtype, two mutants yielded no longer progeny with the original mutant phenotype; these mutants were not analyzed further. The remaining 18 mutants were all recessive and were assigned to 15 complementation groups. Based on these numbers, we estimate that, under the growth conditions tested, about 50-100 genes are specifically involved in ascospore formation in A. nidulans . Three mutations were mapped by parasexual analysis: two mutations could be assigned to a specific chromosome, and one was associated with a translocation breakpoint. For all 18 mutants, the contents of the "barren" fruiting bodies were cytologically analyzed. A large proportion of the mutants, namely 11 out of 18, arrested in meiotic prophase I (like uvsC114 ; chapter 4 ) or metaphase I (like bimD6 ; chapter 3 ). It is thus possible that this new collection contains mutants that are specifically affected in meiotic recombination and/or sister chromatid cohesion. We suggest a strategy to clone the corresponding wild-type genes, by selecting for the appearance of prototrophic progeny clones that result from meiotic reassortment of auxotrophic markers.

    In the General Discussion ( chapter 6) , we speculate upon the possible links between sister chromatid cohesion and recombination in mitosis and meiosis. Whereas in mitosis centromeric cohesion primarily serves chromosome disjunction, arm cohesion may play additional roles in repair by recombination. Cohesion complexes at the basis of the chromatin domains/loops may function as nucleation sites for the assembly of recombinational repair complexes and assist these complexes in finding a homologous template in a precisely corresponding segment of the undamaged sister chromatid. They may thus be responsible for the observed bias for the sister chromatid as a template for recombinational repair during mitotic G2. In meiosis I, recombination has to be directed towards a non-sister chromatid of the homologous chromosome and, at the same time, arm cohesion has to be maintained and possibly even reinforced to ensure correct reductional chromosome segregation. Consequently, both the recombinational repair machinery and the cohesion complexes function in meiosis in a modified form. In this modified form, cohesion complexes may serve as a basis for axial element formation. DSBs are probably produced concomitantly with axial element assembly. As we proposed for mitotic recombinational repair, we hypothesize that meiotic DSBs are transferred to the basis of chromatin loops/domains, where they are brought into contact with the meiotic cohesion complexes and additional proteins required for recombinational repair. However, we speculate that homology search on the sister chromatid will now be blocked by linear element components so that another template for homologous recombination has to be found. Furthermore, axial element components, in concert with the modified recombination complex, will possibly establish DNA-DNA contacts with a non-sister chromatid of the homologous chromosome. Thus, by providing the basis for linear element formation and assembly of a meiosis-specific recombination complex, cohesion complexes may contribute to the preference for a non-sister chromatid of the homologous chromosome as a template for homologous recombination in meiosis.

    Evolution of genetic systems in filamentous ascomycetes
    Nauta, M.J. - \ 1994
    Agricultural University. Promotor(en): R.F. Hoekstra. - S.l. : Nauta - ISBN 9789054851998 - 159
    pezizomycotina - genetica - evolutie - fylogenie - oorsprong - fylogenetica - pezizomycotina - genetics - evolution - phylogeny - origin - phylogenetics

    A great variety of genetic systems exist in filamentous ascomycetes. The transmission of genetic material does not only occur by (sexual or asexual) reproduction, but it can also follow vegetative fusion of different strains. In this thesis the evolution of this variability is studied, using theoretical population genetic models.

    First the evolution of different reproductive systems is studied. It is found that homothallism (allowing selfing) most probably evolved from heterothallism. (with two mating types), and that a polymorphism of homo- and heterothallism can be evolutionary stable. A variable fitness of ascospore production is predicted as an explanation for hermaphroditism in heterothallic species and the formation of both asexual and sexual spores by homothallic species.

    Secondly the evolution of vegetative incompatibility (VI) is studied. VI prevents vegetative fusion of different strains, and is very common between different natural isolates. In many species a large number of Vegetative Compatibility Groups (VCGs) is found, that only show vegetative fusion within and not between groups. After a comparison of different selective regimes, it is concluded that a harmful cytoplasmic element offers the most plausible selective explanation for the evolution of VI. However, the effects of genetic drift appear to be important in generating large numbers of VCGs and can override the effects of selection.

    Next, attention is focused on spore killing. This is a form of segregation distortion (or meiotic drive), causing the death of half the number of spores in an ascus. In a model it is found that the evolution of spore killing can only be explained if 'Spore killers' have some additional advantage during the process of killing.

    Finally, a model is presented for the evolution of sexual incompatibility (SI) in Podospora anserina. As the existence of SI cannot be explained on its own, a hypothesis is studied, that explains SI as an anti meiotic drive device. Although the model shows that this hypothesis could be true, experimental evidence is needed to confirm this.

    Bosbranden en het gevaar voor Rhizina undulata
    Gremmen, J. - \ 1976
    Wageningen : De Dorschkamp (Bericht / Rijksinstituut voor Onderzoek in de Bos- en Landschapsbouw "De Dorschkamp" nr. 92)
    pezizomycotina - bosbouw - plantenziekteverwekkende schimmels - plantenziekteverwekkende bacteriën - bomen - bosbranden - bosschade - rhizina undulata - pezizomycotina - forestry - plant pathogenic fungi - plant pathogenic bacteria - trees - forest fires - forest damage - rhizina undulata
    Inwendige ontsmetting van door Ascochyta pisi aangetaste erwtezaden met de antibiotica rimocidine en pimaricine, benevens enkele aspecten van het parasitisme van deze schimmel
    Dekker, J. - \ 1957
    Wageningen University. Promotor(en): A.J.P. Oort. - Wageningen : Veenman - 80
    plantenziekteverwekkende schimmels - vicia - gewasbescherming - fungiciden - pezizomycotina - zaadbehandeling - zaden - desinfectie - immunologie - antigenen - plant pathogenic fungi - vicia - plant protection - fungicides - pezizomycotina - seed treatment - seeds - disinfection - immunology - antigens
    One of the plant diseases, which is still difficult to control, is leaf, stem and pod spot of peas, caused by the fungus Ascochyta pisi. Seed disinfection is unsatisfactory, because the fungus may penetrate deep into the seed, beyond the reach of superficially active disinfectants. Therefore a search was made for disinfectants, penetrating the seeds, using ascochyta-infected seeds for tests. The most effective compounds were the newly discovered antibiotics rimocidin and pimaricin, products of Streptomyces spp. They penetrated the seeds and eliminated internal fungus without impairing germination. An 18-h soaking reduced the percentage of seeds with viable mycelium from 80 to less than 1 %. Dry or almost dry treatment was less effective. A further drawback of these antibiotics was the reversible loss of fungicidal activity inside plant tissue through adsorption, and inactivation by UV radiation.
    Rimocidin and pimaricin were nevertheless of interest because of their selectivity, being highly toxic to most fungi, but hardly toxic to bacteria and higher plants. Remarkable also was ready uptake by plants and seeds despite their large complex molecules.

    A new aspect of the parasitism of this fungus was that the fungus was often present in plant parts or whole plants without symptoms. Rarely the fungus was even isolated from seeds, harvested from plants without symptoms. It was also observed that during ripening of the pods many pycnidia appeared outside the limited brown-edged spots, typical for this disease.
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