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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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    Antimicrobial peptides with therapeutic potential from skin secretions of polyploid frogs of the Pipidae family
    Mechkarska, M.P.M. - \ 2013
    Wageningen University. Promotor(en): Jerry Wells. - [S.l.] : s.n. - ISBN 9789461735508 - 224
    kikkers - pipidae - secreties - antimicrobiële peptiden - gastheer-pathogeen interacties - frogs - pipidae - secretions - antimicrobial peptides - host pathogen interactions

    The emergence of pathogenic bacteria and fungi resistant to commonly used antibiotics poses a serious threat to public health and necessitates novel treatment approaches in order to control infections. Antimicrobial peptides (AMPs) are one of the central components of the system of innate immunity and due to their non-specific and highly destructive mechanism of killing, pathogens will develop resistance at lower rates than conventional antibiotics.Skin secretions of frogs from the family Pipidae are a rich source of AMPs which show potential for development into therapeutic agents.

    Until recently, the only representatives of the Pipidae family frogs from which dermal AMPs had been identified were the diploid frog Silurana tropicalis, the tetraploid frog Xenopus laevis and the octoploid frog Xenopus amieti. Therefore, this program of research was undertaken with the aim to isolate, purify and characterize AMPs with therapeutic potential from skin secretions of other polyploid species of African clawed frogs of the Pipidae family. Emphasis is given to the application of the AMPs as markers to elucidate the taxonomic relationships and evolutionary history of the frogs. The study also investigates the effects which polyploidization and interspecies hybridization have had on the multiplicity of AMPs in frog skin secretions.

    Chapter 2 and Chapter 3 present data from the peptidomic analysis of norepinephrine-stimulated skin secretions of two well-characterized and closely related tetraploid Xenopus species – X. borealis and
    X. clivii. Multiple peptides with varying degrees of antimicrobial activity were isolated. Structural characterization demonstrated that they were orthologous to magainins, peptide glycine-leucine-amide (PGLa), caerulein-precursor fragments (CPFs) and xenopsin-precursor fragments (XPFs), previously isolated from S. tropicalis, X. laevis and X. amieti. CPF-B1 and CPF-C1 were the most abundant AMPs in the skin secretions of X. borealis and X. clivii respectively. CPF-B1 (GLGSLLGKAFKIGLKTVGKMMGGAPREQ) was active against clinical isolates of the hospital-associated pathogens, methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Acinetobacter baumannii (MDRAB) with MIC = 5 µM and showed low hemolytic activity against human erythrocytes (HC50 >200 µM). CPF-C1 (GFGSLLGKALRLGANVL.NH2) also showed potent activity against a range of Gram-negative bacteria. CPF peptides, therefore, represent promising candidates for development into therapeutic agents for use against these emerging antibiotic-resistant pathogens.

    The genera Silurana and Xenopus are united in the subfamily Xenopodinae and have a complex evolutionary history. Chapter 4 includes data from the peptidomic analysis of skin secretions from an incompletely characterized tetraploid species termed “S. new tetraploid 1”with chromosome number 2n=40 and the octoploid species X. andrei (2n=72). The species represent model systems in which to study of the fate of duplicated AMP genes following putative allopolyploidization events. Multiple peptides belonging to the PGLa, XPF, and CPF familes were identified. The primary structures of the AMPs from X. andrei indicate aclose phylogenetic relationship between this species and the previously studied X. amieti. Three CPF peptides from “S. new tetraploid 1” showed potent, broad-spectrum antimicrobial activity and are present in high abundance. In contrast, only a single CPF peptide was isolated in low yield from the X. andrei secretions. There is no increase in the multiplicity of the AMPs in skin secretions of “S. new tetraploid 1”and the octoploid X. andrei when compared to the diploid
    S. tropicalis (2n=20) and the tetraploid X. laevis (2n=36). It is concluded that nonfunctionalization (gene silencing) has been the most common fate of duplicated AMP genes following polyploidization in the Silurana and Xenopus lineages.

    AMPs constitute a characteristic ‘‘fingerprint’’ of a particular frog species that may be used for an unequivocal taxonomic classification. Two populations of the tetraploid X. muelleri, occupying separate non-contiguous ranges in east and west Africa, are studied in Chapter 5. Their taxonomic relationship is unclear and it has been proposed that the western population represents a separate species referred to as
    X. muelleri West while the eastern population retains the original name X. muelleri. A comparison of the primary sequences of AMPs in skin secretions reveals that no orthologous peptide from the two populations of X. muelleri has the same amino acid sequence. Additionally, the X. muelleri secretions, like those from X. clivii, did not contain a PGLa peptide whereas the X. muelleri West secretions contained two members of this family. The data indicate that X. muelleri West is more closely related to X. borealis than to X. muelleri and so provide strong support for the proposal that X. muelleri and
    X. muelleri West should be described as separate species.

    In contrast to species in the subfamily Xenopodinae, frogs from the subfamily Pipinae have not been investigated as a source of AMPs. The AMP profile in skin secretions from Hymenochirus boettgeri as a representative of genus Hymenochirus (subfamily Pipinae) is described in Chapter 6. A novel family of five structurally-related peptides, designated as hymenochirins, was identified. Hymenochirin-1B (IKLSPETKDNLKKVLKGAIKGAIAV AKMV.NH2) is C-terminally α-amidated whereas hymenochirins-2B - 5B have the general structure XKIPX2VKDTLKKVAKG X2SX2 AGAX3.COOH. The most abundant peptide in the secretions was hymenochirin-3B (IKIPAVVKDTLKKVAKGVLSAVAGALTQ). Synthetic replicates of hymenochirin-1B - 4B possess broad-spectrum antimicrobial activity and relatively weak hemolytic activity and so represent potential candidates for development into therapeutically valuable agents against drug-resistant pathogens. The hymenochirins show very low structural similarity with the antimicrobial peptides isolated from skin secretions of S. tropicalis and X. laevis consistent with the proposed ancient divergence of the Pipinae and Xenopodinae.

    The F1 hybrid frogs X. laevis x X. muelleri represent a model of interspecies hybridization in the Pipidae family that does not result in an increase in ploidy. They arestudied in Chapter 7 and the data obtained provide an insight into the mode of inheritance of AMPs. A total of 18 different AMPs were isolated from skin secretions of the female hybrids. In addition to the complement of AMPs from the parent species, three previously undescribed peptides (magainin-LM1, PGLa-LM1, and CPF-LM1) were purified from the secretions of the hybrid frogs that were not detected in secretions from either X. laevis or X. muelleri. Magainin-LM1 differs from magainin 2 from X. laevis by a single amino acid substitution (Gly13 ®Ala) but PGLa-LM1 and CPF-LM1 differ appreciably in structure from orthologs in the parent species. CPF-LM1 shows potent, broad-spectrum antimicrobial activity and is hemolytic. The data indicate that hybridization increases the multiplicity of host-defense peptides in skin secretions. As the female F1 hybrids are fertile, hybridization may represent an adaptive strategy among Xenopus species to increase protection against pathogenic microorganisms in the environment.

    The thesis is completed by a general discussion in Chapter 8 of theresults and conclusions in Chapters 2-7. The potential of AMPs from skin secretions of frogs belonging to the Pipidae family is reviewed from three different aspects: promising candidates for development into therapeutic valuable anti-infective agents; reliable taxonomic and phylogenetic markers; and tools to study the fate of duplicated genes in Xenopus and Silurana. The interspecies Xenopus hybrids are proposed as a suitable model to perform future studies on the mode of inheritance of skin AMPs.

    Pyrethrum secondary metabolism: biosynthesis, localization and ecology of defence compounds
    Ramirez, A.M. - \ 2013
    Wageningen University. Promotor(en): Harro Bouwmeester; Marcel Dicke, co-promotor(en): Maarten Jongsma. - [S.l.] : s.n. - ISBN 9789461735171 - 187
    tanacetum cinerariifolium - pyrethrinen - metabolisme - biosynthese - verdedigingsmechanismen - plantenfysiologie - afweersecreties - secreties - tanacetum cinerariifolium - pyrethrins - metabolism - biosynthesis - defence mechanisms - plant physiology - defensive secretions - secretions

    The use of botanical insecticides is in today’s world an attractive alternative to the less safe and environmentally malign synthetic chemicals, whose overall longer persistence in the environment does not only make them more contaminant, but also increases their chances of causing a rapid development of resistance in the target pest and having negative-side effects on beneficial organisms. Yet, at present only a handful of botanical products are in commercial use for insect control on crops. Among the most important botanical insecticides (pyrethrins, rotenone, neem and essential oils), pyrethrins have the longest history of effective use against a wide range of insects and best record of low toxicity to mammals. Although pyrethrins were relegated from their once prominent position in the mid-1930’s, the recent market trends towards “reduced risk” pesticides and host plant resistance have brought pyrethrins back to the attention and initiated the generation of knowledge around them. Pyrethrins refer to an oleoresin extracted from the dried daisy-like flowers of pyrethrum (Tanacetum cinerariifolium). The active constituents are six esters formed by a combination of two acids (chrysanthemic acid and pyrethric acid) and three alcohols (pyrethrolone, cinerolone and jasmolone, collectively called rethrolones).The esters of chrysanthemic acid with the rethrolones constitute type I pyrethrins, whereas the esters of pyrethric acid are collectively known as type II pyrethrins. Apart from being the source of pyrethrins, pyrethrum also produces a range of other defense compounds collectively known as sesquiterpene lactones, which have also been implicated in plant defense against herbivores, pathogens, and competing plant species. Although pyrethrins and sesquiterpene lactones are found throughout the whole plant, the highest concentrations of both types of compounds are found in the achenes of the flowers, which are densely covered with glandular trichomes. GC-MS analysis revealed that trichomes of mature achenes contain sesquiterpene lactones and other secondary metabolites, but no pyrethrins. Although glandular trichomes were known to participate in the production of mono- and sesquiterpene compounds that were stored in or emitted from the subcuticular cavity just outside the apical cells, here we demonstrate that basipetal secretion can also occur. In pyrethrum, the monoterpene-derived portion of pyrethrins, chrysanthemic acid (CA), is translocated from the trichomes to the pericarp, where it is esterified into pyrethrins that accumulate in the intercellular space. We also show that during seed maturation, pyrethrins stored in the pericarp are absorbed by the developing embryo, and that during seed germination these embryo-stored pyrethrins are recruited by the germinating seedling, which, due to the lack of trichomes, cannot produce defense compounds themselves. At early stages, not only sesquiterpene lactones that diffuse to the soil from the seed coat, but also the pyrethrins found in the seedlings, seem to play a more important role as antimicrobials than as insecticides.

    Although there has been considerable progress on the chemistry of pyrethrins, the molecular/biochemical basis of their biosynthesis was largely unknown. The acid and alcohol moieties of pyrethrins derive from distinct pathways. Whereas the alcohol portion is believed to be derived from linolenic acid and share a large part of its biosynthetic pathway with jasmonic acid, the acid moieties are monoterpenes with a cyclopropane ring that are supposed to be derived from an irregular monoterpene pathway. Before the start of this project, only one enzyme, chrysanthemyl diphosphate synthase (TcCDS), had been isolated and demonstrated to catalyze the first step in the biosynthesis of the acid portion of pyrethrins, which consist of the condensation of two molecules of DMAPP to produce chrysanthemol (COH) via chrysanthemyl diphosphate. During this project an additional acyltransferase enzyme (TcGLIP) was isolated by another group and demonstrated to be responsible for the esterification of (1R,3R)-chrysanthemoyl-CoA and (S)-pyrethrolone, one of the last steps in the biosynthesis of pyrethrins, which was demonstrated in this thesis to likely take place in the pericarp.

    To identify additional genes of the pyrethrin biosynthetic pathway, we generated three EST libraries derived from ovaries, trichomes and leaves. Gene candidates were obtained either by keyword interrogation of the annotated contigs or by blasting the libraries with known genes catalyzing similar reactions in other plants. Given the likelihood of cytochrome P450s as potential candidates to catalyze the missing steps in the biosynthesis of the acid moiety of pyrethrins, the pyrethrum EST libraries, were first interrogated for genes encoding cytochrome P450 (CYP) enzymes with a developmental expression pattern similar to TcCDS, and a specific expression in CA-producing glandular trichomes. Experiments with yeast microsomes allowed the selection of two enzymes capable of converting COH into chrysanthemal. Although after agro-infiltration none of these enzymes affected the background level of CA, one of them (Ct21854) resulted in a strong reduction of COH emission, which correlates with a significantly higher amounts of a CA conjugate, confirming that Ct21854 qualifies as a chrysanthemic acid synthase efficiently converting COH into CA.

    Rethrolones have been proposed to originate from linolenic acid and share part of the oxylipin pathway with jasmonic acid, which in turns implies that one of the first committed steps should involve a lipoxygenase enzyme, catalyzing the hydroperoxidation of linolenic acid at position 13 of the hydrocarbon chain. Based on this assumption the pyrethrum EST libraries were interrogated for genes encoding LOX enzymes. The expression patterns of twenty-five lipoxygenase EST contigs were characterized, and the ones with a developmental regulation similar to TcCDS and TcGLIP were selected. Subsequently, the molecular cloning of a lipoxygenase, TcLOX1, was carried out. Recombinant TcLOX1was demonstrated to catalyze the peroxidation of the linolenic acid substrate at the C13

    position. The gene shares the developmental and trichome-specific expression pattern with TcCDS, suggesting that a trichome production and translocation could be operating for the alcohol moiety of pyrethrins as well.

    Finally, besides pyrethrins, pyrethrum plants are also a source of sesquiterpene lactones. Even though considerable information on bioactivity and industrial significance of pyrethrum sesquiterpene lactones is available, the localization and biosynthetic origins were largely unknown. Like in other species of the Asteraceae family, pyrethrum sesquiterpene lactones are exclusively stored in trichomes and it is shown that germacratrien-12-oic acid (GAA) is most likely the central precursor of all known sesquiterpene lactones found in pyrethrum. Candidate genes implicated in the first two committed steps leading from farnesyl diphosphate to GAA were retrieved from the pyrethrum trichome EST library, cloned, and characterized in yeast and in planta. Furthermore, a gene encoding an enzyme capable of catalyzing the C6 hydroxylation of GAA was characterized. This hydroxylation results in spontaneous lactonization likely resulting in the putatively identified C6-C7-costunolide-derived STLs in pyrethrum. However, the enzyme may also catalyze the hydroxylation at the C6 position of the lactonized precursor of all reported C7-C8-type STLs.

    In conclusion, the work compiled in this thesis presents new insights into the participation of pyrethrum trichomes in the biosynthesis and selective trafficking of pyrethrins precursors and STLs in opposite directions, and contributes to understanding the role of these flower-stored secondary metabolites in the immunization of the next generation against insect and fungal pathogens. Moreover, this study makes an important contribution towards understanding the biochemical and molecular bases of pyrethrins and STLs, the two most relevant bioactive compounds found in pyrethrum.

    Ongevleugelde lieveheersbeestjes
    Lommen, S. ; Kuik, A.J. van - \ 2012
    Bomen, het vakblad voor de boomverzorging (2012)21. - p. 14 - 17.
    straatbomen - aphidoidea - plantenplagen - organismen ingezet bij biologische bestrijding - coccinellidae - adalia - honingdauw - secreties - bestrijdingsmethoden - onderzoek - street trees - aphidoidea - plant pests - biological control agents - coccinellidae - adalia - honeydew - secretions - control methods - research
    Bladluizen in stadsbomen zorgen soms voor grote overlast. Vooral onder lindebomen is er jaarlijks wel een periode van honingdauwoverlast. Sommige steden gaan het probleem te lijf met het uitzetten van tweestippelige lieveheersbeestjes in de lindebomen. Het resultaat is niet altijd bevredigend. Nu is het effect van deze maatregel voor het eerst onderzocht. Voor dit onderzoek is echter speciaal gebruikgemaakt van een in de natuur voorkomend ongevleugeld type van dit lieveheersbeestje. De verwachting is dat dit langer in de boom blijft en daardoor beter is in bladluisbestrijding.
    Plant - Microbiele Brandstofcel (MFC): exudate productie : het optimaliseren van wortelexudatie met een split-root systeem
    Khodabaks, M. ; Blok, C. ; Berg, C.C. van den; Snel, J.F.H. - \ 2009
    Bleiswijk : Wageningen UR Greenhouse Horticulture - 13
    akkerbouw- en tuinbouwbedrijven - kassen - exudaten - wortelexudaten - secreties - aminozuren - organische zuren - micro-organismen - organische stof - suikers - koolhydraten - teelt onder bescherming - microbiële brandstofcellen - glastuinbouw - biobased economy - crop enterprises - greenhouses - exudates - root exudates - secretions - amino acids - organic acids - microorganisms - organic matter - sugars - carbohydrates - protected cultivation - microbial fuel cells - greenhouse horticulture
    De plant microbiële brandstofcel of Plant Microbial Fuel Cell (Plant$MFC) is een technologie die het op basis van een nieuw principe mogelijk maakt direct elektriciteit of biofuels aan een plant te onttrekken, zonder dat deze geoogst hoeft te worden (Strik en Helderman, 2004). Levende planten zetten door fotosynthese zonne-energie om in energiehoudende biomassa zoals eiwitten, suikers, zetmeel, cellulose en ligine. Van de netto vastgelegde koolstof wordt doorgaans een fractie van 40 tot 60 % naar de wortels getransporteerd. Van de hoeveelheid koolstof getransporteerd naar het wortelstelsel wordt door planten een fractie van 50 tot 70 % uitgescheiden naar de bodem in oplosbare vorm (exudaten en secreties). Deze exudaten en secreties bestaan onder andere uit suikers, aminozuren, organische zuren en koolhydraten welke gemakkelijk door micro-organismen kunnen worden omgezet. De uitgescheiden organische stof kan deels door natuurlijk voorkomende micro-organismen worden omzet in electriciteit. Als deze electriciteit in de een of ander vorm wordt opgevangen en benut is sprake van een MFC. In een MFC is het zaak het aandeel en de activiteit van de electriciteit producerende micro-organismen hoog te maken en te houden
    SELDI-TOF-MS of saliva : Methodology and pre-treatment effects
    Schipper, R.G. ; Loof, D. ; Groot, J. de; Harthoorn, L.F. ; Dransfield, E. ; Heerde, W. van - \ 2007
    Journal of Chromatography. B, Analytical technologies in the biomedical and life sciences 847 (2007)1. - ISSN 1570-0232 - p. 45 - 53.
    flight mass-spectrometry - human whole saliva - 2-dimensional gel-electrophoresis - liquid-chromatography - protein-components - proteomic analysis - identification - histatins - peptides - secretions
    Interest in saliva as a diagnostic fluid for monitoring general health and for early diagnosis of disease has increased in the last few years. In particular, efforts have focused on the generation of protein maps of saliva using advanced proteomics technology. Surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is a novel high throughput and extremely sensitive proteomic approach that allows protein expression profiling of large sets of complex biological specimens. In this study, large scale profiling of salivary proteins and peptides, ranging from 2 to 100 kDa was demonstrated using SELDI-TOF-MS. Various methodological aspects and pre-analytical variables were analysed with respect to their effects on saliva SELDI-TOF-MS profiling. Results show that chip surface type and sample type (unstimulated versus stimulated) critically affect the amount and composition of detected salivary proteins. Factors that influenced normal saliva protein profiling were matrix composition, sample dilution and binding buffer properties. Delayed processing time experiments show certain new peptides evolving 3 h post-saliva donation, and quantitative analyses indicate relative intensity of other proteins and peptides changing with time. The addition of protease inhibitors partly counteracted the destabilization of certain protein/peptide mass spectra over time suggesting that some proteins in saliva are subject to digestion by intrinsic salivary proteases. SELDI-TOF-MS profiles also changed by varying storage time and storage temperature whereas centrifugation speed and freeze¿thaw cycles had minimal impact. In conclusion, SELDI-TOF-MS offers a high throughput platform for saliva protein and peptide profiling, however, (pre-)analytical conditions must be taken into account for valid interpretation of the acquired data.
    Endogenous cellulases in stylet secretions of cyst nematodes
    Smant, G. - \ 1998
    Agricultural University. Promotor(en): J. Bakker; A. Schots; F.J. Gommers. - S.l. : Smant - ISBN 9789054859260 - 111
    plantenparasitaire nematoden - globodera rostochiensis - heterodera glycines - secreties - gastheer parasiet relaties - speeksel - cellulase - plant parasitic nematodes - globodera rostochiensis - heterodera glycines - secretions - host parasite relationships - saliva - cellulase
    This thesis describes the identification ofβ-1,4-endoglucanases (cellulases) in stylet secretions of the two cyst nematodes species, Globodera rostochiensis and Heterodera glycines . A novel method was developed to raise monoclonal antibodies that were directed to subventral oesophageal gland secretions. These monoclonal antibodies were used to characterise and to immunopurify two secretory proteins. Partial sequence data from these proteins enabled the cloning of two homologous genes from each of the two cyst nematode species.

    The predicted amino acid sequences revealed a high similarity with bacterial cellulases, whereas no homology was found with eukaryotic cellulases. Evidence is provided for the endogenous origin the nematode cellulases that may have been acquired from bacteria by horizontal gene transfer.

    In vitro sensitivity and tolerance of Fusarium solani towards chitinases and [beta]-1,3-glucanases
    Sela - Buurlage, M.B. - \ 1996
    Agricultural University. Promotor(en): P.J.G.M. de Wit, co-promotor(en): L.S. Melchers. - S.l. : Sela-Buurlage - 211
    plantenziekteverwekkende schimmels - deuteromycotina - gewasbescherming - planten - dieren - secreties - fytoalexinen - parasitisme - fysiologie - gastheer parasiet relaties - tuberculariaceae - plant pathogenic fungi - deuteromycotina - plant protection - plants - animals - secretions - phytoalexins - parasitism - physiology - host parasite relationships - tuberculariaceae

    In agriculture, fungal diseases have always been one of the major problems. Many options exist to combat the pathogens responsible. Application of fungicides is for specific diseases a very effective means of control. However, new strains of fungal pathogens may emerge showing resistance to such compounds. Moreover, environmental and health concerns have made these chemicals less favourable. Crop rotation is a possibility to control disease, but is economically less attractive for farmers. Traditional plant breeding to obtain resistant cultivars fits best in a system of sustainable agriculture. However, this technology is very laborious and time consuming. Also, desired resistance traits might not be available within the species or even within related species. Since the development of technology for genetic engineering of plants, new strategies for introducing resistance in plants to fungal pathogens have emerged.

    In the first chapter of this thesis, a review is presented on the various strategies that are used or could possibly be used in the future to genetically engineer fungal resistance. One of the strategies followed at MOGEN involves overexpression of one or more antifungal proteins. The work, presented in this thesis, is part of this strategy. An in vitro assay had been established to assist in the isolation and identification of such antifungal proteins (Woloshuk et al., 1991) and has played a pivotal role in the results described here. In search for such antifungal proteins, the phenomenon of induced resistance is exploited. Nicotiana tabacum, cv. Samsun NN, when inoculated with tobacco mosaic virus (TMV), acquires resistance to subsequent pathogen attack. Synthesis of a large number of pathogenesis-related (PR) proteins is induced (Linthorst, 1991).

    In Chapter 2 results are described using protein extracts from tobacco leaves inoculated with TMV. These induced extracts were calibrated for the levels of known PR-proteins and tested in vitro on a variety of fungi. The majority of fungi were inhibited in growth by these extracts. Spores of all fungi were far more sensitive to induced protein extracts if pregerminated before addition of the extracts, when compared to assaying without pregermination.

    The natural location of many antifungal tobacco PR-proteins, such as Chi-I, Glu-I and AP24, is the vacuole. However, since many pathogens reside in the intercellular spaces, overexpression of these proteins is expected not to yield the desired protective effect. Therefore, genes were modified in such a way that proteins, in stead of being targeted to the vacuole, were rerouted extracellularly. Results of these experiments are presented in Chapter 3.

    In Chapters 4 and 5 several of the tobacco PR-proteins were purified and assayed for their in vitro antifungal effects. In Chapter 4, the proteins of group PR-2, β-1,3-glucanases, and PR-3, chitinases, were assayed for their antifungal activity, either alone or in synergy. Apoplastic 5, the isolation, enzymatic activity and antifungal activity of the class I PR-4 CBP20, is described.

    In Chapter 6, the proteins from transgenic plants described in Chapter 4, were reisolated in order to analyze whether extracellular targeting had affected antifungal activity.

    As observed in Chapter 2, non pregerminated fungal spores were far less sensitive to induced protein extracts compared to germlings. In Chapters 7 and 8, the phenomenon of decreased sensitivity occurring during incubation with antifungal proteins is further investigated using F.solani f.sp. phaseoli as a model system. The effect of gerniination time before addition of proteins was studied. Results presented indicate that macroconidia adapt to the presence of specific chitinases only during the first three hours of germination. Concomitantly, as described in Chapter 8, specific protease(s) are released by the germinating spore capable of cleaving the chitin-binding domain from Chi-I, and CBP20. The influence of this chitin-binding domain on the level of antifungal activity of Chi-I, and CBP20 as well as its role on the adaptation phenomenon has been determined.

    The overall results described in this thesis are summarized in Chapter 9. The use of an in vitro assay to assist in the isolation of antifungal proteins is addressed in detail. Since it was demonstrated that macroconidia can adapt to the presence of specific antifungal proteins, the relevance of this observation is discussed. Finally, the importance of in vitro identification of antifungal proteins in engineering fungal resistant plants is demonstrated.

    Sensory and nutritional effects of amino acids and phenolic plant compounds on the caterpillars of two Pieris species
    Loon, J.J.A. van - \ 1988
    Agricultural University. Promotor(en): L.M. Schoonhoven. - S.l. : Van Loon - 210
    aminozuren - dieren - voedingsgedrag - insecten - larven - Lepidoptera - fenolen - fytoalexinen - plantenplagen - gewasbescherming - planten - secreties - fenolverbindingen - rupsen - rhopalocera - amino acids - animals - feeding behaviour - insects - larvae - Lepidoptera - phenols - phytoalexins - plant pests - plant protection - plants - secretions - phenolic compounds - caterpillars - rhopalocera

    The relationships between caterpillars of Pierisbrassicae L. and Pierisrapae L. (Lepidoptera: Pieridae) and a common host plant Brassicaoleracea L. were studied using chemosensory and nutritional techniques. Attention was focussed on amino acids, which are in part essential nutrients, and on phenolic and flavonoid derivatives of two aromatic amino acids, that are products of the secondary metabolism in the host plant.

    An electrophysiological study of amino acid gustation showed that in both species 14 out of 22 amino acids were stimulants to a receptor cell in a maxillary sensillum. The nutritionally essential amino acids were generally stronger stimuli than dispensable ones. A correlation analysis provided indirect evidence that the amino acid receptor possessed four sites, one less specific and three or possibly four specific ones. A comparison of data on free amino acid concentrations in B.oleracea with dose-response relations of the amino acid cell showed that this cell can quantitatively sense foliar amino acids.

    Phenolic acids and an anthocyanin that naturally occur in B.oleracea elicited neural responses from two to three maxillary gustatory cells. Chlorogenic and protocatechuic acids, both carrying ortho-substituted hydroxyl groups on the aromatic ring, were the most effective stimulants. A steep increase in responsiveness was occurring with increasing concentrations in the range 0.2 - 5.0 mM. P.rapae was the less sensitive of both species. Flavonols were ineffective. The predominant anthocyanin in B.oleracea , cyanin, evoked neural activity in some cells but inhibited the activity in gustatory cells sensitive to sugars, amino acids and glucosinolates in P.brassicae . Chemosensory responsiveness was reflected in preference behaviour. Naturally occurring levels of phenolic acids in B.oleracea as found in phytochemical studies are able to affect sensory processes in the caterpillars (Chapter 3).

    Assessment of possible metabolic effects of dietary phenolics and some other dietary variations was performed using a flow-through respirometer. This was designed to monitor continuously the gas exchange of feeding caterpillars during the complete final instar. The results of these measurements were compared to the results obtained using standard gravimetric techniques that make use of the measurement of food intake to calculate metabolic efficiency. Respirometric results yielded small effects on the energetic efficiency of growth, which was in contrast to gravimetric results. The causes of the discrepancies between both methods and the consequences of these findings for studies on insect food utilization in general are discussed (Chapter 4).

    The nutritional utilization of amino acids and nitrogen was studied comparatively for caterpillars of both species on an artificial diet and on B.oleracea . Food consumption in the final instar was lower on the artificial diet. More food was consumed when leaf amino acid content was lower. Relationships were found between food consumption and the absorption efficiencies of most of the essential amino acids. Absorption efficiencies for glycine, cystein and serine were lower on the artificial diet, differences for other amino acids were small between the diets. Amino acid utilization patterns were similar for both species. Balance sheet calculations showed that an extensive conversion from phenylalanine to tyrosine occurred. For both species indications were obtained that tyrosine and cystein may become limiting for growth when dietary protein levels are low (Chapter 5).

    Phenolic acids (caffeic and chlorogenic acids) and flavonoids (oenin and quercetin-3-rutinoside) inhibited survival, development and growth when larvae of both species were continuously exposed to these compounds present in an artificial diet. P.brassicae was distinctly more sensitive at lower levels of the compounds (0.4 and 1.0 mM). Final instars of both species were much less sensitive than earlier instars. Growth inhibition in final instars was primarily due to reduced food consumption. The results suggest a potential role of phenolic acids and flavonoids, normal constituents of leaves of B.oleracea , in defence against Pieris caterpillars (Chapter 6).

    Seven cultivars of B.oleracea were offered as food to study their suitability as a host plant for larvae of both caterpillar species. Parameters of larval performance showed differences between cultivars. Highperformance liquid chromatography was used to analyse leaf tissues of five cultivars with respect to concentrations of phenolic acids and flavonoids. Each of the cultivars was found to have its own quantitative pattern of these compounds. Unidentified polar flavonoid components were detected in highly variable amounts. These preliminary results further support a potential role of phenolic and flavonoid compounds in resistance of B.oleracea against Pieris (Chapter 7).

    Callus and cell culture of Tagetes species in relation to production of thiophenes
    Ketel, D.H. - \ 1987
    Agricultural University. Promotor(en): J. Bruinsma; B. de Groot. - Wageningen : Ketel - 130
    dieren - asteraceae - celkweek - chemische analyse - chemische samenstelling - meristemen - fytoalexinen - gewasbescherming - planten - secreties - thiofeen - weefselkweek - tagetes - animals - asteraceae - cell culture - chemical analysis - chemical composition - meristems - phytoalexins - plant protection - plants - secretions - thiophene - tissue culture - tagetes
    The production of thiophene-biocides by cell cultures invitro was simultaneously investigated with Tageteserecta , T.patula and T.minuta . The calli from which the liquid cultures had to be derived differed between species in the appearance of organoid structure, texture, and colour, Independently of the nutrition of the plants and explants. In particular, the difference between the friability of calli of different species Is obviously related to the expression of the activity of silent genes In only a late phase of the callus andlor the cell suspension culture. Therefore 'origin effects' may eventually determine the suitability of calli to Initiate liquid cultures (Chapter 1). The differences between calli, however, showed that the production of thiophenes In the calli was positively related with the measure of differentiation. Rapidly growing and fine granular cell sus~ pensions, for Instance obtained from smooth calli of T.minuta , did not produce thiophenes (Chapters II and III).

    Differentiated calli of T.erecta did not provide suitable material to initiate cell cultures In liquid medium. However, minced cauliflower-like calli of T.patula with irregularly occurring small root- or shoot-like differentiations, formed large cell aggregates (3- 8 mm) in liquid media. These cell aggregates accumulated non-polar thiophenes and released spontaneously relatively high amounts of a water-soluble thiophene (BBTOH) in to the medium (Chapter VII). Apparently, the Increased morphological dedifferentation of calli runs parallel with a decreased production of thiophenes In the cell suspensions derived from them. The long-term accumulation of thiophenes In cell aggregates and the release of such compounds into the mediom open perspectives for the commercial production of such compounds under fermentor conditions.

    Embedding of the fine granular suspension cells of T.minuta in alginate resulted in the release of secondary metabolites into the liquid medium, but did not provide adequate conditions to reinitiate the production of thiophenes (Chapter V). In contrast, naturally formed cell aggregates which can be considered as a natural system of entrapment, as formed by T.patula cells, obviously provide suitable conditions for the production of thiophenes.

    Genetic transformation of intact Tagetes by means of infection with wild-type and mutant strains of Agrobacteriumtumefaciens and A . rhizogenes , induced neoplastic outgrowth of various organized and unorganized tissues without added growth regulators (Chapter VI). The change in this potential may be related to an altered synthesis of endogenously formed phytohormones. The species-dependent relationship between morphological differentiation and thiophene production persisted In all transformed tissues examined.

    In conclusion, the results of the present experiments on thiophene production in cell cultures of Tagetes species support the view that, despite the totipotency of plant cells (Chapter IV), major differences exist between closely related species in the ability to serve as a biotechnological unit invitro . Consequently, extensive research to adapt a certain recalcitrant plant species for plant cell biotechnology should be avoided by looking for a better producing species.

    Een modelsynthese voor momilactonen : onderzoek naar de totaalsynthese van 9betaH-pimara-7,15-dienen
    Sicherer - Roetman, A. - \ 1984
    Landbouwhogeschool Wageningen. Promotor(en): Æ. de Groot. - Wageningen : Sicherer-Roetman - 167
    gewasbescherming - planten - dieren - secreties - fytoalexinen - lipiden - biosynthese - diterpenoïden - sesquiterpenoïden - terpenen - etherische oliën - sesquiterpenen - modellen - onderzoek - plant protection - plants - animals - secretions - phytoalexins - lipids - biosynthesis - diterpenoids - sesquiterpenoids - terpenoids - essential oils - sesquiterpenes - models - research

    This thesis describes investigations into the total synthesis of momilactones, germination inhibitors and phytoalexins isolated from rice. These compounds possess a Δ 7,8 -pimaradiene type skeleton with an unusual trans-syn ring-arrangement (figure 1).

    In chapter 1 a survey is given of the momilactones and the other hitherto known trans-syn pimaranelactones, with emphasis on their structures, biosynthesis and physiological activities.

    Chapter 2 is devoted to a literature survey of synthetic studies towards trans-syn(-cis) perhydrophenanthrene systems. The chemical reactivity of trans-syn pimaranelactones is also discussed.

    In chapter 3 the results are presented of a synthetic investigation, based on the Diels-Alder reaction depicted in scheme 1.

    This approach provided a total synthesis of Δ 8,9 -pimaradiene and Δ 8,9 -sandaracopimaradiene. However, attempts to isomerize the double bond to the desired Δ 7,8 -position met with little success. We therefore turned our attention towards starting compounds bearing an oxo group on C-7, in order to utilize this group for the introduction of the Δ 7,8 -double bond at a later stage.

    Using the work of W.L.Meyer and coworkers as a starting point, we stereospecifically synthesized a trans-syn-cis perhydrophenanthrene system as outlined in scheme 2. This part of the investigations is described in chapter 4. Several ways for the stereospecific introduction of a second substituent on C-13 were investigated. our synthesis of compound 159 could probably have been elaborated further, but we chose to focus our attention on a more promising approach which is described in chapter 5.

    Our stereospecific synthesis of trans-syn-cis perhydrophenanthrene systems, which forms the subject of chapter 5, is based on the stereospecific Diels-Alder reaction depicted in scheme 3 and culminates in the succesful synthesis of model compound 122. Initially we used 2- trimethylsilyloxybutadienes as diene components, but severe hydrolysis problems were encountered with the resulting adducts. These problems were effectively overcome by using diene 190. The adduct possesses a regiospecific silylenolether system which can be alkylated at C-13. Adduct 191 could be deformylated and stereoselectively reduced to the alcohol 197 leaving the t-butyldimethylsilylenolether intact. Two possible synthetic routes were then investigated.

    Alkylation of compound 199 with 2-ethoxy-1,3-dithiolan surprisingly only gave one thiolanyl compound (200) which proved to have the thiolanylgroup in the α-position. Reduction and hydrolysis of this compound gave the hydroxyaldehyde 205. However, during the Wittig reaction of the latter compound, equilibration occurred via (retro-)aldol reaction, resulting in considerable epimerization at C-13. Only a small amount of α-vinylproduct was found. Oxidation and Wolff-Kishner reduction finally afforded the model compound 122.

    Alkylation of compound 201 with 2-ethoxy-1,3-dithiolan yielded stereospecifically the β-thiolanyl product, as could be expected for steric reasons. This product was elaborated further as shown. Here, too, a (retro-)aldol reaction occurred during the Wittig reaction of compound 225, resulting in both hydroxy-epimers of the β-vinyl alcohol. No α-vinylproduct could be detected in this case. This concluded the stereospecific synthesis of compound 122.

    X-ray crystallography of thiolanyl-compounds 200 and 219 and 13 C-NMR spectroscopy were used to establish the stereochemistry of a number of reaction products, especially concerning the configuration at C-13. Details of these measurements can be found in chapter 6.

    Finally, in chapter 7, the results of the investigations are summarized and evaluated in relation to the total synthesis of momilactones.

    Influence of different compositae on population density of Pratylenchus penetrans and some other root - infesting nematodes
    Hijink, M.J. ; Winoto Suatmadji, R. - \ 1967
    Wageningen : [s.n.] (Mededeling / Laboratorium voor phytopathologie Serie nemat., no. 50) - 12
    dieren - asteraceae - bestrijdingsmethoden - cultuurmethoden - heteroderidae - geïntegreerde bestrijding - geïntegreerde plagenbestrijding - fytoalexinen - plantenziekten - plantenplagen - gewasbescherming - planten - pratylenchus - secreties - tylenchidae - animals - control methods - cultural methods - integrated control - integrated pest management - phytoalexins - plant diseases - plant pests - plant protection - plants - secretions
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