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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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    Mining into interspecific bacterial interactions
    Tyc, Olaf - \ 2016
    Wageningen University. Promotor(en): Wietse de Boer, co-promotor(en): Paolina Garbeva. - Wageningen : Wageningen University - ISBN 9789462578340 - 234
    soil bacteria - secondary metabolites - microbial interactions - antibiotics - nutrients - bodembacteriën - secundaire metabolieten - microbiële interacties - antibiotica - voedingsstoffen

    In terrestrial ecosystems bacteria live in close proximity with many different microbial species and form complex multi-species networks. Within those networks bacteria are constantly interacting with each other and produce a plethora of secondary metabolites like antibiotics, enzymes, volatiles and other compounds from diverse chemical classes. Several independent studies revealed that the production of secondary metabolites by soil bacteria can be influenced by the interaction with other microorganisms in their vicinity.

    In this thesis we show how interspecific interactions between soil bacteria influence the production of soluble and volatile secondary metabolites, gene expression and fitness. To elucidate the effect of interspecific interactions on antimicrobial activity in soil bacteria a high-through-put screening method was developed and applied on a collection of 146 rhizobacterial isolates obtained from similar habitats. In addition we examined if the production of volatile organic compounds is influenced by interspecific interactions. Thus, the identity and antimicrobial activity of volatiles produced by bacteria cultivated in monoculture as well in interaction were examined. Furthermore a sand microcosm approach was applied to investigate how Pseudomonas fluorescens strain Pf0-1 responded to the presence of monocultures and mixtures of a Gram-negative (Pedobacter sp. V48) and a Gram-positive (Bacillus sp. V102) bacterial strain under two nutritional conditions.

    The interaction between a gram-negative Burkholderia and a gram-positive Paenibacillus isolate was subjected to detailed metabolome, volatolome and transcriptome analysis. One distinct volatile and one non-volatile compound produced only during interspecific interaction but not in the monoculture were identified. The activity of the interacting bacteria and the compounds produced during interaction were tested against a range of human and plant pathogens.

    In summary, this thesis extends the knowledge about the effect of interspecific bacterial interactions on secondary metabolites production (soluble and volatiles), gene expression and fitness in bacteria. The exploitation of such bacterial interspecific interactions can be an important “tool” for the discovery of novel antimicrobial and agro-chemical compounds. The obtained knowledge can help in selecting the right players in synthetic communities that fulfil important ecosystem services like disease suppression in agricultural crop systems.

    The secondary metabolome of the fungal tomato pathogen Cladosporium fulvum
    Griffiths, S.A. - \ 2015
    Wageningen University. Promotor(en): Pierre de Wit; Pedro Crous, co-promotor(en): Jerome Collemare. - Wageningen : Wageningen University - ISBN 9789462575813 - 167
    passalora fulva - secundaire metabolieten - metabolomen - genen - genomica - biologische activiteit - biosynthese - natuurlijke producten - passalora fulva - secondary metabolites - metabolomes - genes - genomics - biological activity - biosynthesis - natural products

    Secondary metabolites (SMs) are biologically active organic compounds that are biosynthesised
    by many plants and microbes. Many SMs that affect the growth, behaviour or survival of other
    organsisms have been re-purposed for use as medicinal drugs, agricultural biocides and animal
    growth promoters. The majority of our anti-infective and anti-cancer drugs are currently derived
    from Streptomyces, bacteria that are free living, filamentous, and ubiquitous in terrestrial habitats.
    Genome sequencing and mature in silico approaches to genome mining has revealed that filamentous
    fungi contain very large numbers of genes related to SM production. Yet these genes are typically
    silent under laboratory conditions. There are now many tools and strategies available to activate
    or clone silent SM genes. This thesis details our efforts to apply various methods to define and
    then manipulate SM genes in Cladosporium fulvum, a biotrophic pathogenic fungus of tomato
    containing many silent SM genes and gene clusters.

    In chapter 1, the relevance of SMs to medicine and agriculture is considered. Filamentous fungi
    are presented as untapped sources of potential useful SMs, as their genomes are often rich in SM
    biosynthetic genes that are silent under most conditions. Methods to activate these silent genes and
    increase the chemical diversity of fungi are detailed. These include the deletion or over-expression
    of genes encoding regulatory proteins, the use of chemical inhibitors, and the manipulation
    of growth conditions. Heterologous expression of silent SM genes in a production host is also
    discussed as a tool for bypassing host regulatory mechanisms altogether. C. fulvum is introduced
    as an organism that has been intensively studied as a biotrophic plant pathogen. Genomic analysis
    showed that this fungus has twenty-three core SM genes, a large catalogue composed of 10
    polyketide synthases (PKSs), 10 non-ribosomal peptide synthases (NPS), one PKS-NPS hybrid
    and one dimethylallyl tryptophan synthase (DMATS). Transcriptional profiling showed that the
    majority was silent during growth on tomato and in vitro. Cladofulvin is introduced as the sole
    detectable SM produced by C. fulvum during growth in vitro. This presented an opportunity to
    apply the aforementioned strategies to induce these silent genes and obtain new compounds. The
    importance of cladofulvin and structurally related anthraquinones are briefly discussed as potential
    medicines. The value of the cladofulvin biosynthetic gene cluster is also emphasised as a potential
    source of novel biosynthetic enzymes.

    In chapter 2 the SM gene catalogue identified during the analysis of the C. fulvum genome was
    analysed in further detail. Each locus containing a core SM gene was inspected for other biosynthetic
    genes linked to SM production, such as those encoding decorating enzymes and regulators. Products
    of these SM genes or gene clusters were speculated, based on their similarity to those characterized
    in other fungi. Six gene clusters were located in the genome of C. fulvum that are conserved in other
    fungal species. Remarkably, two predicted functional gene clusters were linked to the production
    of elsinochrome (PKS1) and cercosporin (PKS7), toxic perylenequinones that generate reactive
    oxygen species (ROS). We profiled the expression of core SM genes during the growth of C. fulvum
    under several in vitro conditions. Expression of each core SM gene was measured by RT-qrtPCR
    and the resulting SM profile was determined by LC-MS and NMR analyses. Confirming previous
    findings, the majority of SM genes remained silent and only cladofulvin was detected. During
    growth on tomato only two core genes, PKS6 and NPS9, were clearly expressed, but both were
    significantly down-regulated during colonization of the mesophyll tissue of tomato leaves. We
    confirmed that cladofulvin does not cause necrosis on solanaceous plants when infiltrated into
    their leaves. In contrast to other biotrophic fungi that have a reduced SM production capacity, our
    studies of C. fulvum suggest that down-regulation of SM biosynthetic pathways might represent
    another mechanism associated with a biotrophic lifestyle.

    In chapter 3 our efforts to activate cryptic pathways in C. fulvum are described, with the aim
    of discovering new compounds. Many Ascomycete-specific global regulators of SM production
    and morphological development in other fungi were identified in C. fulvum. We investigated
    three intensively studied regulators, VeA, LaeA and HdaA. Deleting or over-expressing the genes
    encoding these regulators in C. fulvum yielded no new detectable SMs. Cladofulvin biosynthesis
    was strongly affected by each regulator; HdaA is an activator while VeA and LaeA are repressors of
    cladofulvin production. Attempts were made to stimulate SM production in the mutants and wild
    type strains by growing them on different carbon sources, but only cladofulvin biosynthesis was
    affected. Interestingly, cladofulvin production was stimulated by carbon limitation and strongly
    repressed in the presence of saccharose. Similar to observations made in other fungi, the deletion of
    VeA or LaeA did not affect viability, but maturation and conidiation were affected. Sporulation was
    not overtly affected by the loss of HdaA, but Δhdaa deletion mutants did not produce cladofulvin.
    This suggests that cladofulvin production is not required for asexual reproduction. The main
    finding of this chapter is that global regulator manipulation cannot considered to be a universal
    tool to discover new fungal natural products.

    In chapter 4, anthraquinones and closely related compounds such as anthrones, anthracyclines
    and xanthones are considered. Emodin is perhaps the most well characterised anthraquinone that
    is produced by many fungi and plants. Once synonymous only with constipation, this former
    laxative has since been investigated for its useful anti-cancer, anti-diabetic, anti-infective and antiinflammatory properties. Cladofulvin is a homodimeric anthraquinone composed of nataloe-emodin joined in a remarkably asymmetrical configuration. Dimeric anthraquinones and xanthones are also bioactive, most commonly tested for anti-infective and anti-cancer activities. Despite the ubiquity and medicinal qualities of anthraquinones and related compounds, very few of their biosynthetic pathways are known. No enzymes capable of dimerizing anthraquinones had yet been identified. In this chapter we demonstrated that cladofulvin was very cytotoxic towards human cancer cell-lines, crucially, up-to 300-fold more than its monomeric precursor nataloe-emodin against certain celllines. This became an added incentive to elucidate the cladofulvin pathway and identify the enzyme responsible for dimerizing nataloe-emodin. We confirmed earlier predictions that PKS6/claG is the core gene which starts cladofulvin biosythesis. Deletion of claG abolished cladofulvin production
    and no related metabolites were observed. A route to cladofulvin biosynthesis was proposed, guided
    by the work performed on the monodictyphenone biosynthetic pathway in Aspergillus nidulans.

    We predicted early acting cladofulvin genes and cloned them for heterologous expression in A.
    oryzae strain M-2-3. Using this approach we were able to confirm the first five genes in cladofulvin
    biosynthesis, claBCFGH, which yielded a reduced and dehydrated form of emodin. This is the
    point at which the pathways to cladofulvin and monodictyphenone production diverge. It was
    speculated that this emodin-related intermediate might be converted into nataloe-emodin by claK
    and/or claN. Finally, it was confirmed that the final step in the cladofulvin pathway is encoded by
    claM. Targeted deletion of claM yielded a mutant that accumulated nataloe-emodin and emodin
    but no cladofulvin. We discuss how the sequence of claM and ClaM will accelerate the discovery
    of functionally similar genes and enzymes, providing a template to engineer enzymes capable of
    forming novel dimers from existing monomers.

    In chapter 5 the natural role of cladofulvin was considered. This SM is consistently produced by
    C. fulvum and global regulator mutants in vitro. The respective biosynthetic genes appear most
    active during early and late stages of infection of tomato, but are down-regulated during biotrophic
    growth phase (chapter 2). The Δclag mutants (chapter 3) were not overtly different from the wild
    type during growth in vitro. We inoculated tomato plants with this mutant in order to test whether
    or not cladofulvin was required for normal infection. Simultaneously, we inoculated a C. fulvum
    transformant carrying an extra copy of the cladofulvin pathway-specific relulator, OE.claE, fused
    to the promoter region of the Avr9 effector gene. The strain was expected to produce cladofulvin
    once the fungal hyphae penetrate host stomata and begin to colonise the apoplastic space. In this
    way, we aimed to test the effect of cladofulvin over-production on disease symptom development.
    The growth of each strain on tomato plants was monitored by RT-qrtPCR at 4, 8 and 12 days post
    inoculation (dpi). At each time point the infections were inspected microscopically to detect any
    phenotypic abnormalities. We report that the loss of claG did not result an abnormal infection.
    Both wild type and ΔclaG mutants sporulated without causing necrosis or dessication of host leaves.
    In distinct contrast, brown spots appeared on leaves infected by the OE.claE transformant between
    8 – 12 dpi. This was accompanied by much stronger fungal growth and significant accumulation
    of cladofulvin. The leaves became desiccated and brittle before the fungus conidiated. Possible
    reasons for this phenotype are discussed. A small suite of in vitro experiments was performed on the
    Δclag and wild type strains in order to test the role of cladofulvin in survival. Consistent with the
    absence of a photoprotective pigment, Δclag spores were considerably more sensitive to ultraviolet
    (UV) radiation. Suggesting a role in protection against low temperatures, Δclag spores were less
    resistant to repeated cycles of freezing and thawing. Cladofulvin biosynthesis was stimulated and
    repressed by cold and heat shocking mature C. fulvum colonies, respectively. Altogether, these
    results suggested that cladofulvin confers resistance to abiotic stress.

    In chapter 6 the results obtained in this thesis are discussed in a broader context. Particularly,
    the discovery of the cytochrome P450 that is involved in dimerization of anthraquinones might
    enable discovery of homologous genes encoding enzymes with different specificities. Combining
    bioinformatic and functional analyses should prove to be a powerful strategy for discovering
    compounds with new biological activities, or enzymes relevant to metabolic engineering.

    Weerbaarheid van de plant wordt langzamerhand ontrafeld : Op zoek naar de juiste balans tussen weerbaarheid en productie (interview met Jantineke Hofland-Zijlstra)
    Kierkels, T. ; Hofland-Zijlstra, J.D. - \ 2014
    Onder Glas 11 (2014)5. - p. 42 - 43.
    glastuinbouw - gewasproductie - sierplanten - stressfactoren - verdedigingsmechanismen - secundaire metabolieten - optimalisatiemethoden - landbouwkundig onderzoek - gewasbescherming - plantgezondheid - greenhouse horticulture - crop production - ornamental plants - stress factors - defence mechanisms - secondary metabolites - optimization methods - agricultural research - plant protection - plant health
    Met een goede klimaatbeheersing kan de teler Botrytis in de hand houden. Maar eigenlijk is de weerbaarheid van de plant bij deze schimmel veel belangrijker. Bij een hogere weerbaarheid hoef je veel minder scherp op het klimaat te letten en bespaar je op het middelengebruik. Diverse onderzoekinstellingen werken aan meer inzicht.
    Breeding for pepper fruit quality: a genitical metabolomics approach
    Wahyuni, Y. - \ 2014
    Wageningen University. Promotor(en): Raoul Bino, co-promotor(en): Arnaud Bovy; E. Sudarmonowati; A.R. Ballester. - Wageningen : Wageningen University - ISBN 9789461739582 - 193
    capsicum annuum - capsicum frutescens - capsicum chinense - capsicum baccatum - plantenveredeling - metabolomica - gewaskwaliteit - secundaire metabolieten - gezondheid - genetische kartering - rijp worden - capsicum annuum - capsicum frutescens - capsicum chinense - capsicum baccatum - plant breeding - metabolomics - crop quality - secondary metabolites - health - genetic mapping - ripening

    A diverse collection of 32 pepper accessions was analysed for variation in health-related metabolites, such as carotenoids, capsaicinoids, flavonoids and vitamins C and E. For each of the metabolites analysed, there was a lot of variation among the accessions and it was possible to identify accessions with high amounts of specific metabolites. While all accessions contained high levels of vitamin C, accession C. chinenseAC2212 was found to be an excellent source of vitamin E, whereas C. annuumLong Sweet accumulated high levels of the flavonoid quercetin. A genetical metabolomics approach was used to study the genetic basis of metabolic traits in a segregating F2 population based on a cross between two contrasting pepper genotypes. This led to the identification of several metabolites QTL hotspots. The genetic basis for the accumulation of several flavonoids in pepper fruit was further investigated, by combining metabolic, gene expression and candidate gene-based marker data. This provided valuable insight into the key genes important for flavonoid accumulation in pepper fruit. The results of this study will help breeders to assist future breeding programs aimed at optimizing the levels of nutritional compounds in pepper fruit.

    Discovery and reconstitution of the secoiridoid pathway of Catharanthus roseus
    Dong, L. - \ 2014
    Wageningen University. Promotor(en): Richard Immink, co-promotor(en): Sander van der Krol. - Wageningen : Wageningen University - ISBN 9789461738462 - 201
    catharanthus roseus - secoïridoïden - secundaire metabolieten - medicinale eigenschappen - genen - biosynthese - catharanthus roseus - secoiridoids - secondary metabolites - medicinal properties - genes - biosynthesis

    Terpene indole alkaloids (TIAs) are important plant-produced secondary metabolites for

    humans, because of their anti-cancer properties. The production of TIAs still fully relies on

    extraction from medicinal plants like Catharanthus roseus, which only contains extreme low

    amounts of these compounds and new ways need to be found to efficiently produce these

    anticancer drugs at low cost. The common precursor for TIAs is strictosidine and in my PhD

    project I tried to produce strictosidine in fast-growing tobacco by transferring the genes of the

    whole biosynthesis pathway into tobacco. At the onset of my project 6 out of the presumed

    12 genes of the pathway in C. roseus had not been discovered yet. My thesis tells the story of

    discovery and characterization of the missing genes and reconstruction of the full strictosidine

    pathway in tobacco.

    1

    Genetics of insect resistance to plant defence
    Vermeer, K.M.C.A. - \ 2014
    Wageningen University. Promotor(en): Marcel Dicke, co-promotor(en): Peter de Jong. - Wageningen : Wageningen University - ISBN 9789461738363 - 199
    insect-plant relaties - co-evolutie - planten - plaagresistentie - verdedigingsmechanismen - insectenplagen - secundaire metabolieten - phyllotreta nemorum - barbarea vulgaris - genetisch bepaalde resistentie - genetische analyse - insect plant relations - coevolution - plants - pest resistance - defence mechanisms - insect pests - secondary metabolites - phyllotreta nemorum - barbarea vulgaris - genetic resistance - genetic analysis

    Plants are chemically defended against insect herbivory in various ways. They produce a broad range of secondary metabolites that may be toxic or deterrent to insects. Specialist insects, however, are often capable of overcoming these defences. The yellow striped flea beetle (Phyllotreta nemorumL.) is a specialist that feeds on crucifers (Brassicaceae) such as Sinapis arvensisand Barbarea vulgaris. In Denmark, two types of Barbarea vulgarisvar. arcuataare distinguished: one with pubescent leaves (P-type) and one with glabrous leaves (G-type). All individuals of P. nemorumcan feed on B. vulgarisP-type. Barbarea vulgarisG-type, on the other hand, is chemically defended against most P. nemorumindividuals during the flea beetle reproductive season. The defence compounds are hypothesized to be saponins, a class of compounds with various biological effects and insecticidal properties. Despite high levels of these saponins during summer, some flea beetles can and do feed on B. vulgaris G-type. The ability of P. nemorumto feed on B. vulgarisG-type is heritable; resistance against the defence of B. vulgarisG-type is controlled by dominant major resistance genes (R-genes). One dominant R-allele of an R-gene is enough to convert a susceptible beetle into a resistant one. Despite knowledge of the inheritance patterns of resistance in the flea beetles, which have been demonstrated to be variable, the underlying mechanism of flea beetle resistance has, so far, remained unclear. This prompted me to investigate, as an initial part of my thesis, the genetic basis of the flea beetle adaptation to the defence of B. vulgarisG-type.

    The interaction between B. vulgarisand the flea beetle is a unique natural model system to study chemical defences in plants and counter-adaptations in insects. Plant and insect are both polymorphic with respect to the trait involved in resistance and hereby provide an excellent opportunity to study the geographic aspects of the evolution of the resistance trait in both interacting species. In this thesis, I focus on the resistance of the flea beetle, and take the presence of different genotypes of the plant as a given. Phyllotreta nemorumis a major pest, for example in oil seed rape. Understanding how resistance evolves in P. nemorumwill not only benefit flea beetle control, but also control of other pest insects. Understanding insect resistance includes knowledge of seasonal, geographic and genetic variation in both plant defense and herbivore adaptation.The R-gene has a remarkable distribution. Flea beetle populations living on B. vulgarisG-type consist solely of resistant individuals, but on host plant patches nearby B. vulgarisG-type lower frequencies of resistant beetles are found than one would expect with the amount of gene flow found at the neutral level between these subpopulations.

    The aim of this thesis was to find the gene that is held responsible for the resistance of P. nemorumto the defences of B. vulgaris, investigate the distribution of this resistance trait and explain the distribution of this trait in natural populations. The following questions were addressed: (1) what is the genetic basis of the adaptation under study? (2) how is the resistance distributed across flea beetle populations in Denmark? and (3) which factors underlie this distribution?

    In order to answer these questions, I used an integrated approach. I have combined a candidate gene approach (CHAPTER3) with an empirical approach via the study of variation in resistance in flea beetle populations (CHAPTER4), and a population genomics approach by using molecular markers to gain insight in the genomic make-up of the population and its connection with the resistance trait (CHAPTERS5 and 6). The population genomics approach is a recent advance in methods to detect the involvement of selection in the distribution of alleles at presumably adaptive loci. Using this approach one can distinguish locus-specific effects, like directional selection, from genome-wide effects, on the distribution of alleles at loci of interest.

    The population genomics approach is introduced in CHAPTER2 together with the Geographic Mosaic Theory of Coevolution. I illustrate how processes underlying this theory of coevolution can be investigated with the population genomics approach. According to the geographic mosaic theory of coevolution, reciprocal selection between interacting species only happens in so-called hot-spots. Hot spots can be identified using population genomics and genetic variation found at specific loci can be attributed to locus-specific processes such as directional selection. For the B. vulgaris- flea beetle system this means that with a population genomics approach we can examine whether the distribution of resistant flea beetles on alternative host plants is only influenced by migration, or also by selection (CHAPTER5). Another valuable utility of the population genomics approach is to investigate whether a candidate gene for the R-gene is under selection, by looking whether a candidate gene is experiencing locus-specific effects beside genome-wide effects when comparing flea beetle populations living on B. vulgarisG-type with populations living on alternative host plants (CHAPTER6).

    However, before using a population genomics approach to compare the resistance trait or a candidate gene with parts of the genome that only experience genome-wide effects, I have tried to identify the genetic basis of the flea beetle adaptation to the defence ofB. vulgarisG-type. In CHAPTER3, I have addressed this question by using a candidate gene approach to examine the involvement of a possible detoxifying enzyme in P. nemorum. Genes coding for β-glucosidase were a candidate for genes underlying the difference between resistant and susceptible beetles, because β-glucosidase is used as detoxifying enzyme by other organisms resistant to saponin defence. Three different β-glucosidase cDNA sequences were cloned from Danish flea beetle lines. We named them β-glucosidase A, B and C. β-glucosidase C was only found in resistant lines and not in the susceptible line. We then tested if recombinant β-glucosidase C breaks down the most abundant and most effective defence compound in B. vulgarisG-type, hederagenin cellobioside. β-glucosidase C was able to deglycosylate one glucose unit of hederagenin cellobioside, when expressed in an insect cell line. This suggests that expressed β-glucosidase C can deglycosylate antifeedant saponins and may play a role in the resistant flea beetle’s ability to overcome the defence of B. vulgaris. Next, a segregating family was created in which offspring differed in resistance genotype. Again β-glucosidase cDNA sequences were cloned to find a difference in the presence of these β-glucosidases between resistant and susceptible individuals. This time cDNA sequences of β-glucosidases A, B and C were present in both resistant and susceptible individuals although significantly fewer β-glucosidase C cDNA sequence variants were found in susceptible individuals than in resistant individuals. Thus, the genetic basis of flea beetle resistance remains unclear. Further investigation is needed to explore if the β-glucosidase C protein is also capable of inactivating hederagenin cellobioside by hydrolysizing the second glucose unit from the saponin and if there is a difference in enzyme activity of β-glucosidase C between resistant and susceptible beetles.

    Subsequently, in CHAPTER4 I have investigated whether the frequency of resistant beetles decreased in populations living on other host plant patches than B. vulgarisG-type and whether the change in frequency was significant within the flea beetle season. I found that the frequency of resistant beetles varied significantly among years, but there was no evidence for a decrease in the frequency of resistant beetles, the latter being expected if selection acts against the resistance on other host plants than B. vulgarisG-type. Furthermore, I found that the frequency of resistant beetles varied significantly within a flea beetle season. This study demonstrates that relative frequencies of different resistance phenotypes of P. nemorumon other host plants than B. vulgarisG-type are highly dynamic, both within and across years. It is, therefore, important to sample season-wide when one wants to monitor the changes in frequencies of insect resistance in natural systems.

    In CHAPTERS5 and 6 I took a population genomics approach to investigate if the observed geographicaldistribution of resistance of P. nemorumto chemically defendedB. vulgarisin flea beetle populations could be explained by factors that are solely associated with genome-wide effects, such as migration, or also by locus-specific factors like selection at the resistance locus. First, neutral microsatellites were used to reveal the genetic differentiation at parts of the genome that are only influenced by genome-wide processes. Next, the level of neutral genetic differentiation was compared with the genetic differentiation found for the resistance trait. The resistance trait was an outlier in pairwise comparisons between flea beetle populations on B. vulgarisand S. arvensis, meaning that the level of genetic differentiation was significantly higher than expected if the resistance trait experiences only genome-wide effects. The resistance trait was also an outlier in the pairwise comparison between populations on S. arvensis, which suggests that the resistance trait is also under directional selection on other host plants than B. vulgarisG-type.

    Additionally, I examined in CHAPTER6 if the homologous β-glucosidases B and C sequences found in CHAPTER3 correspond to two alleles of the major resistance gene, because of their similarity and their presence in flea beetle lines. The sequence of β-glucosidases C had so far only been found in resistant individuals, so we hypothesized it to be the dominant resistance allele and the sequence of β-glucosidases B would then correspond to the susceptible allele. In order to find out if this hypothesized PneR-gene (Phyllotreta nemorum R-gene) is the resistance gene, we first directly compared resistance phenotypes of beetles collected from populations on B. vulgarisG-type and S. arvensiswith genotypes derived with primers developed for β-glucosidase B and C. The phenotype of the flea beetles did not match the genotype derived with the β-glucosidase primers. Additionally, the frequency of heterozygotes and homozygotes of the PneR-gene genotype was not significantly deviating from Hardy-Weinberg Equilibrium which implies that there are no locus-specific effects involved when both sequences are seen as one gene with two alleles. A population approach was taken like in CHAPTER5, this time including the genetic differentiation estimated for the candidate gene as well. The candidate gene behaved similar to the neutral loci while the resistance trait was an outlier in most pairwise comparisons between flea beetle populations. If both sequences are alleles of the same gene, then the candidate gene is not directly responsible for the flea beetle resistance to B. vulgarisG-type defence.

    The results presented in this thesis show the complexity of genetic processes (either genome-wide or locus specific) affecting local adaptation and the distribution of a resistance trait in insects in natural populations. Furthermore, the present study shows that when studying coevolution between insect and host plant by means of adaptive traits, also geographical and seasonal variation in allele frequencies should be considered. A multidisciplinary approach to study adaptation in plant-insect interactions such as used in this thesis, will benefit research on plant-insect interactions, including applied research such as studying the potential of host plants as dead-end traps for pest insects and preventing/diminishing the development of resistance by pest insects to crop defences.

    The ecology of plant secondary metabolites : from genes to global processes
    Iason, G.R. ; Dicke, M. ; Hartley, S.E. - \ 2012
    Cambridge [etc.] : Cambridge University Press (Ecological reviews ) - ISBN 9780521157124 - 335
    secundaire metabolieten - planten - plantensamenstelling - verdedigingsmechanismen - ziekteresistentie - gastheer parasiet relaties - ecologie - genen - biosynthese - herbivoren - secondary metabolites - plants - plant composition - defence mechanisms - disease resistance - host parasite relationships - ecology - genes - biosynthesis - herbivores
    Plant secondary metabolites (PSM) such as terpenes and phenolic compounds are known to have numerous ecological roles, notably in defence against herbivores, pathogens and abiotic stresses and in interactions with competitors and mutualists. This book reviews recent developments in the field to provide a synthesis of the function, ecology and evolution of PSM, revealing our increased awareness of their integrative role in connecting natural systems. It emphasises the multiple roles of secondary metabolites in mediating the interactions between organisms and their environment at a range of scales of ecological organisation, demonstrating how genes encoding for PSM biosynthetic enzymes can have effects from the cellular scale within individual plants all the way to global environmental processes. A range of recent methodological advances, including molecular, transgenic and metabolomic techniques, are illustrated and promising directions for future studies are identified, making this a valuable reference for researchers and graduate students in the field.
    Hoge kilogramopbrengst gaat niet altijd samen met hoge kwaliteit
    Heuvelink, E. ; Kierkels, T. - \ 2007
    Onder Glas 4 (2007)5. - p. 54 - 55.
    groeimodellen - plantenontwikkeling - plantenfysiologie - gewaskwaliteit - stressfactoren - stress - maximum opbrengst - relatie tussen groei en oogst - milieubeheersing - kwaliteitszorg - secundaire metabolieten - glastuinbouw - growth models - plant development - plant physiology - crop quality - stress factors - stress - maximum yield - growth yield relationship - environmental control - quality management - secondary metabolites - greenhouse horticulture
    Samengaan van hoge productie en een goede kwaliteit vergt drie stappen. Ten eerste moet de fotosynthese optimaal zijn. Ten tweede is een goede verdeling van de assimilaten naar nuttige delen van de plant belangrijk. De derde stap is per gewas anders. Soms is het nodig af te zien van de hoogste productie om een goede kwaliteit te krijgen. Bij tomaat geeft een hogere EC een betere smaal maar minder productie. Bij roos zou continue groeilicht veel meer opbrengst geven, maar bij de consument verwelkt de bloem dan zeer snel. Bij veel gewassen is enige stress niet verkeerd. Bloemisterijgewassen zijn dan sterker; groenten smaken soms beter. In Wageningen wordt getracht nieuwe groeimodellen te maken die tuinder en veredelaar bijstaan in het streven naar meer interne kwaliteit
    Metabolomics voor de selectie van resistentie en een verbeterde biologische bestrijding
    Jongsma, M.A. ; Bouwmeester, H.J. ; Krens, F.A. - \ 2005
    chrysanten - rassen (planten) - tomaten - secundaire metabolieten - insecten - plantenziekteverwekkers - geurstoffen - ziekteresistentie - plagenbestrijding - gewasbescherming - chrysanthemums - varieties - tomatoes - secondary metabolites - insects - plant pathogens - odours - disease resistance - pest control - plant protection
    Het geurprofiel van 25 verschillenden chrysantenrassen en 10 verschillende tomatensoorten werd geanalyseerd en gecorreleerd met resistentiegegevens tegen trips en witte vlieg
    Cultivation of Marine Sponges: From Sea to Cell
    Sipkema, D. - \ 2004
    Wageningen University. Promotor(en): Hans Tramper, co-promotor(en): Rene Wijffels; Ronald Osinga. - Wageningen : Detmer Sipkema - ISBN 9789085040743 - 184
    secundaire metabolieten - farmaceutische producten - sponsen - kweektechnieken - secondary metabolites - pharmaceutical products - sponges - culture techniques
    Marine sponges are one of the richest natural sources of secondary metabolites with a potential pharmaceutical application. A plethora of chemical compounds, with widely varying carbon skeletons, possessing among other anticancer, antiviral, antibiotic, antiinflammatory and antimalaria activity has been discovered. While for most metabolites their molecular mode of action is still unclear, for a substantial number of compounds the mechanisms by which they interfere with the pathogenesis of a wide range of diseases has been reported. Knowledge on the mode of action is one of the key factors required to transform bioactive compounds into medicines. The rich diversity in bioactive compounds from sponges has provided molecules that interfere with the pathogenesis of a disease at many different points, which increases the chance of developing selective drugs against specific targets (Chapter 2).Unfortunately, these secondary metabolites are usually present in trace amounts, and natural stocks are too small to sustain the development of widely available medicines. The development of ways to obtain large quantities of the secondary metabolites is therefore currently the most important quest. A number of biotechnological methods could potentially provide the required amount of bioactive substances. Three methods were studied in this thesis:

    Ex situculture

    The term ex situ culture refers to cultivation of functional sponges outside of the sea. One of the crucial issues for the ex situ cultivation of sponges is the design of a suitable growth medium. Generally sponges are regarded as particle feeders (bacteria and algae), but they are also capable of the uptake of (partly) dissolved organic carbon sources. The use of powdered substrates can be beneficial for the ex situ culture of sponges under controlled conditions, because an optimal mix of nutrients can be developed and a constant quality can be guaranteed. The ex situ growth rates of sponges cultured on these substrates could be improved, when compared to the sea, but they remain low and resulted in long-term experiments. In order to optimise the growth rate of sponges, it is important to have insight in the way that sponges grow. The suitability of three different models (linear, exponential and radial accretive growth) to describe the growth of both globose and encrusting sponges was assessed. For both morphological appearances, radial accretive growth was the preferred model to simulate the growth. The model can be a valuable tool to make a sound comparison between growth rates of different sponges. In addition, it can be used to study the quantitative effect of factors, such as pressure, light, current, age, temperature or the nutrient source or -concentration on the growth rate of sponges (Chapter 3).

    Primmorphs

    Primmorphs are spherical-shaped sponge-cell aggregates with a diameter of approximately 1 mm. They are formed from a dissociated cell suspension under gentle agitation and resemble buds and gemmules, which are the naturally produced asexual regeneration bodies. Primmorph formation seems to be a universal characteristic of marine sponges, as they were obtained from seven different species. By scanning electron microscopy (SEM) it was observed that the primmorphs are very densely packed sphere-shaped aggregates with a continuous pinacoderm (skin cell layer) covered by a smooth, cuticle-like structure. The latter characteristic is probably the reason why primmorphs are more robust than functional sponges and can be easily maintained for a long time. Incubation of primmorphs in a rich medium to attempt cultivation of the aggregates frequently resulted in the growth of bacterial, fungal and eukaryotic unicellular contaminants, which prevented a growth study of primmorphs. The addition of gentamycin or a mixture of penicillin and streptomycin could usually avoid bacterial contaminants, but eukaryotic contaminants were persistent. The addition of the fungicide amphotericin B or a cocktail of antibiotics (kanamycin, gentamycin, tylosin and tetracyclin) prevented the formation of primmorphs (Chapter 4).

    If primmorphs are actually a kind of experimentally induced regeneration bodies, they could develop into functional sponges. When primmorphs were maintained in seawater enriched with silicate (70 or 150 µM) it was observed that they indeed produced spicules (silica-based skeletal elements) and attached to the bottom of the culture dish, which never occurred at lower silicate concentrations (4 or 25 µM). These results may be explained by available knowledge on the molecular level. Silicate is known to induce the expression of silicatein, the enzyme involved in the production of spicules, at concentrations higher than 60 µM. In addition, silicate has been found to stimulate the biosynthesis of myotrophin, which enhances the production of collagen. Collagen is well known to play an important role in both the attachment of gemmules to a substratum and their subsequent morphogenesis (Chapter 5).

    Sponge-cell culture

    Sponge-cell culture may be the tool to overcome the low growth rate, and the corresponding low production rate of the bioactive metabolites of functional sponges. However, the presence of large numbers of associated bacteria, fungi and unicellular organisms inside sponges has been a major obstacle in the development of sponge-cell lines. They have prevented the formation of axenic sponge-cell suspensions, and proliferating sponge cells in cell cultures were therefore looked at with suspicion.

    For that reason two of prerequisites for the cultivation of sponge cells were developed:

    A method to distinguish sponge cells in culture from contaminants.A method to assess the viability of cells in culture.The 18S rRNA gene is a suitable marker to identify the origin of eukaryotic cells and a genetic detection method based on this gene was developed for the sponge Dysidea avara . The 18S rRNA gene from a Dysidea avara specimen was sequenced and compared to eukaryotic 18S rDNA sequence(s) that were picked up from a proliferating cell culture that originated from a dissociated Dysidea avara specimen. This method proved to be successful to unambiguously detect whether the cells in culture were actually sponge cells or contaminants (Chapter 6).

    Cell viability is an essential tool to study the effect of medium components on cell physiology. Especially in case of primary sponge-cell lines it is important to know whether slow growth is caused by a low specific growth rate or by a low viability of the cells. Trypan blue exclusion is a commonly used method to estimate the viability of cell cultures, but for unknown reasons this does not work properly with sponge cells. Therefore, a flow-cytometric viability assay, based on the combined use of fluorescein diacetate (FDA) and propidium iodide (PI) was developed. The effects of temperature, ammonium and the fungicide amphotericin B on the viability of a primary cell culture were studied as examples to assess the suitability of the test. Cell fluorescence measurements based on incubation of cells with FDA or PI, resulted in a good and reproducible estimate of the viability of primary sponge-cell cultures. It was found that the cells rapidly die at a temperature of 22 °C or higher, but that they are insensitive to ammonium concentrations up to 25 mM. Amphotericin B was found to be toxic to the cells (Chapter 7) and this could explain why no primmorphs were formed in the presence of this antibiotic.

    The current technical status of different methods to produce sponge metabolites was used to study the feasibility of pharmaceuticals from sponges at a large-scale. The production of the metabolites halichondrin B and avarol by chemical synthesis, wild harvest, mariculture, ex situ culture, primmorphs, sponge-cell culture, genetic modification and semi-synthesis were compared on a technical and economical basis, as far as possible. Halichondrin B from a Lissodendoryx sp. and avarol from Dysidea avara were used as model compounds as their products are opposites with respect to their natural concentration inside the sponge. It is concluded that for avarol, which is present in a relatively high concentration, mariculture and ex situ culture could offer feasible methods to compete with currently used medicines against psoriasis. For halichondrin B, the low concentration is a bottleneck for sponge biomass-based production of the compound. A combined approach of (genetically modified) bacterial fermentation (to produce a precursor molecule) followed by a limited number of chemical steps to produce molecules that are derived from sponge chemicals will probably be the most successful method to develop medicines from sponge metabolites that are present in low concentrations (Chapter 8).
    Biosafety of metabolic engineering in plants : literature study in the framework of Policy Program 347, Biological Safety of Transgenic Plants commissioned by the Dutch Ministry of Agriculture, Nature Management and Fisheries
    Nap, J.L. ; Gilissen, L.J.W.J. - \ 2002
    Wageningen : Plant Research International - 46
    secundaire metabolieten - metabolisme - planten - engineering - bioveiligheid - toxicologie - ecologie - pleiotropie - transgene planten - secondary metabolites - metabolism - plants - biosafety - toxicology - ecology - pleiotropy - transgenic plants
    The biosynthesis of sesquiterpene lactones in chicory (Cichorium intybus L.) roots
    Kraker, J.W. de - \ 2002
    Wageningen University. Promotor(en): Æ. de Groot; M.C.R. Franssen; H.J. Bouwmeester. - S.l. : S.n. - ISBN 9789058085313 - 176
    cichorei - biosynthese - sesquiterpenoïde lactonen - secundaire metabolieten - bitterheid - chicory - biosynthesis - sesquiterpenoid lactones - secondary metabolites - bitterness
    Wild chicory(Cichorium intybusL.) is a blue-flowered composite plant that has spread all over the world from the Mediterranean. Sprouts of chicory var.foliosumHegi that are grown in the dark became popular as a vegetable (Belgian endive) halfway through the nineteenth century. Nowadays it is a common crop in Belgium, northern France, and the Netherlands. The well-known bitter taste of chicory is associated with the presence of sesquiterpene lactones of which the three major ones are the guaianolides lactucin, 8-deoxylactucin, and lactucopicrin (Fig. 1). Additionally, smaller amounts of eudesmanolides and germacranolides are present. The average sesquiterpene lactone content of the wild variety(sylvestre) is 0.42% dry weight in the roots and 0.26% in the leaves. The sesquiterpene lactones in chicory act as feeding deterrent toward insects, but may have an antifungal and antibacterial function as well(Chapter1).

    Sesquiterpene lactones are considered as a major class of plant secondary products, which mainly occur in the Asteraceae. Over 4000 different structures are known, but the majority of them has a guaiane, eudesmane, or germacrene framework (i.e. guaianolides, eudesmanolides, germacranolides). Sesquiterpene lactones with such a framework arethought to originate from (+)-costunolide, the most elementary structure of a germacrene sesquiterpene lactone. In this thesis a pathway for the biosynthesis of (+)-costunolide in chicory roots has been established (Fig. 2).

    Figure2. Established pathway for the biosynthesis of(+)-costunolide from farnesyl diphosphate (FPP) in chicory.

    The committed step in the biosynthesis of (+)-costunolide is the cyclisation of farnesyl diphosphate (FPP) into (+)-germacrene A(Chapter2). The involved (+)-germacrene A synthase was isolated from chicory roots and purified 200-fold by a combination of anion exchange and dye-ligand chromatography. The isolated enzyme belongs to the group of sesquiterpene synthases, has aKm-value of 6.6_M, an estimated molecular weight of 54 kD, and a (broad) pH optimum around 6.7. The recent isolation of genes encoding the (+)-germacrene A synthase of chicory makes it possibly to block this crucial step in sesquiterpene lactone biosynthesis, which may result in new less bitter tasting varieties of Belgian endive. Formation of the lactone ring involves the introduction of a carboxylic acid function in the isopropenyl group of (+)-germacrene A(Chapter3). It starts with the hydroxylation of (+)-germacrene A to germacra-1(10),4,11(13)-trien-12-ol by the (+)-germacrene A hydroxylase. This cytochrome P450 enzyme is NADPH-dependent, has a pH optimum at 8.0, and is blue-light reversibly inhibited by CO. Germacra-1(10),4,11(13)-trien-12-ol is subsequently oxidised to germacra-1(10),4,11(13)-trien-12-oic acid via germacra-1( 10),4,11(13)-trien-12-al by pyridine nucleotide dependent dehydrogenases. Some questions about the exact cofactor dependence of the dehydrogenase catalysed reactions remain, but on the whole the best results were obtained with NADP+. Conversion of germacra-1(10),4,11(13)-trien-12-oic acid into (+)-costunolide is catalysed by the (+)-costunolide synthase(Chapter5). This enzyme is also a cytochrome P450 enzyme, since it depends upon NADPH and is blue-light reversibly inhibited by CO. Biosynthesis of (+)-costunolide in the presence of18 O2resulted in the incorporation of one atom of18O. This supports the concept that the lactone ring is formed via a hydroxylation at theC6-position of the germacrene acid, after which the hydroxyl group attacks the carboxyl group atC12. It is not clear whether the final lactonisation is also mediated by the (+)-costunolide synthase or occurs spontaneously (outside the enzyme).
    (+)-Costunolide is converted into 11(S),13-dihydrocostunolide and leucodin by an enzyme extract from chicory roots in the presence of NADPH andO2(Fig. 3). It is to be expected that other sesquiterpene lactones are formed as well in these incubations, but it is unlikely thatthey can be detected by the GC-MS method which was used to analyse the enzyme assays. The formation of 11(S),13-dihydrocostunolide is catalysed by a stereoselective enoate reductase and does also occur in the absence ofO2. The formation of leucodin involves a cytochrome P450 enzyme and presumably also a dehydrogenase, but it is unclear how cyclisation into the guaiane framework takes place. 11(S),13-Dihydrocostunolide is a reasonable intermediate in the biosynthesis of all 11,13-dihydro-sesquiterpene lactones present in chicory, but its involvement in leucodin biosynthesis was not investigated. Notably, leucodin is only one hydroxylation step away from 11(S),13-dihydro-8- deoxylactucin, a minor bitter sesquiterpene lactone of chicory. The germacrene intermediates of sesquiterpene lactone biosynthesis can be isolated from fresh roots ofSaussurea lappa(costus roots)(Chapter4). The occurrence of these germacrene intermediates along with high amounts of (+)-costunolide and dehydrocostus lactone within one and the same plant is an additional proof for the established pathway depicted in Figure 2. The germacrene compounds are susceptible to proton-induced cyclisations and to heat induced Cope rearrangement yielding eudesmanes and elemenes respectively. However, the isolated germacrenes are not that unstable as often suggested by literature. They remain for instance intact during the enzyme incubations at 30°C. The best way to analyse the oxygenated germacrenes is to let them undergo a Cope rearrangement to their corresponding elemenes immediately at the start of the GC-run. This can be achieved by the use of injection port temperatures of at least 250°C; if this is not done they will generally yield very broad peaks(Chapter4). Cope rearrangement into (-)-_-elemene was used to determine the absolute configuration of the enzymatically produced germacrene A on an enantioselective GC-column(Chapter2). In the presence of NADPH, a microsomal pellet from chicory roots is able to hydroxylate various sesquiterpene olefins, which are exogenous to the plant(Chapter6). Most of these hydroxylations take place at the allylic position of an isopropenyl or isopropylidene group (Fig. 4). The number of products obtained from a certain substrate is confined to one or in, a few cases, two sesquiterpene alcohols. Although the microsomal pellet contains various membrane bound enzymes, the majority of hydroxylations is ascribed to the (+)-germacrene A synthase since (+)-germacrene A competitively inhibits their biotransformation. This disputes the common idea that cytochrome P450 enzymes of plant secondary metabolism have a narrow substratespecificity. The unforeseen hydroxylation of (+)-valencene into_-nootkatol is presumably catalysed by a different cytochrome P450 enzyme, possibly the same that is involved in biosynthesis of leucodin from (+)-costunolide. During incubation_-nootkatol is rapidly oxidised further by NAD(P)+-dependent dehydrogenases into nootkatone, a much sought-after component with a distinctive flavour of grapefruit.



    The achieved regioselective, and in the case of_-nootkatol also stereoselective, introduction of a hydroxyl group into sesquiterpene olefins is often difficult to achieve by organic chemical methods. Nonetheless, the small quantities of oxygenated products obtained are a major drawback in the application of the isolated oxidising enzymes from chicory roots. It would be worthwhile to isolate the genes encoding the involved cytochrome P450 enzymes and to functionally overexpress them in yeast. In this way higher enzymatic activities and larger amounts of possibly interesting products may be expected(Chapter7).
    Carvacol krachtig natuurlijk conserveermiddel : trends in ingredienten : dl. 12
    Ultee, A. ; Smid, E. - \ 2000
    Voedingsmiddelentechnologie 33 (2000)12. - ISSN 0042-7934 - p. 11 - 14.
    voedselbewaring - voedselvergiftiging - anti-infectieuze middelen - bacillus cereus - voedingsmiddelen - smakelijkheid - aromatische stoffen - plantaardige oliën - secundaire metabolieten - voedingsmiddelenwetgeving - wetgeving - houdbaarheid (kwaliteit) - opslag - bacteriële toxinen - toxinen - food preservation - food poisoning - antiinfective agents - foods - palatability - flavourings - plant oils - secondary metabolites - food legislation - legislation - keeping quality - storage - bacterial toxins - toxins
    Deze natuurlijke stof fungeert als antioxidant, aroma en als conserveermiddel. Als conserveermiddel is Carvacrol niet toegestaan, wel als smaakstof
    Bactericidal action of carvacrol towards the food pathogen Bacillus cereus : a case study of a novel approach to mild food preservation
    Ultee, A. - \ 2000
    Agricultural University. Promotor(en): F.M. Rombouts; E.J. Smid. - S.l. : S.n. - ISBN 9789058082190 - 96
    voedselvergiftiging - bacillus cereus - voedselbewaring - anti-infectieuze middelen - secundaire metabolieten - etherische oliën - food poisoning - bacillus cereus - food preservation - antiinfective agents - secondary metabolites - essential oils

    A new trend in food preservation is the use of mild preservation systems, instead of more severe techniques such as heating, freezing or addition of chemical preservatives. Carvacrol, a phenolic compound present in the essential oil fraction of oreganum and thyme, is known for its antimicrobial activity since ancient times. This thesis describes a study of the antimicrobial activity of carvacrol towards the foodborne pathogen B. cereus . Carvacrol shows a dose-related inhibition of growth of B. cereus . Concentrations of 0.75 mM and higher inhibit growth completely at 8°C. Below 0.75 mM, carvacrol extends the lag-phase and reduces the specific growth rate as well as the final population density. Exposure to 0.75-3 mM carvacrol decreases the number of viable cells of B. cereus exponentially. Spores are approximately two fold more resistant towards carvacrol than vegetative cells.

    The incubation and exposure temperature have a significant influence on the sensitivity of B. cereus to carvacrol. An increase of the growth temperature from 8°C to 30°C decreases the fluidity of the membrane of vegetative cells and as a consequence, B. cereus becomes less sensitive to carvacrol. The change in membrane fluidity is probably the result of a higher percentage of lower melting lipids in the membranes at 8°C (chemical process) as an adaptation to lower temperature. Cells need to maintain an adequate proportion of the liquid-crystalline lipid in the membrane, as this is the ideal physical state of the membrane. On the other hand, an increase of the exposure temperature from 8 to 30°C, reduces the viability again. This can be explained by an increase of the membrane fluidity at a higher temperature as a result of melting of the lipids (physical process). At a higher membrane fluidity, relatively more carvacrol can dissolve in the membrane and the cells will be exposed to relatively higher concentrations than at a lower membrane fluidity.

    Not only the temperature plays a role in the activity of carvacrol, also pH is an important factor. The sensitivity of B. cereus to carvacrol is reduced at pH 7, compared to other pH-values between pH 4.5 and 8.5.

    Carvacrol interacts with the cytoplasmic membrane by changing its permeability for cations such as K +and H +. Consequently, the dissipation of the membrane potential (Δψ) andΔpH leads to inhibition of essential processes in the cell, such as ATP synthesis, and finally to cell death. At carvacrol concentrations as low as 0.15 mM,Δψis completely dissipated, however the viable count of B. cereus is not affected.

    Vegetative cells of B. cereus can adapt to carvacrol when the compound is present at concentrations below the MIC-value. Compared to non-adapted cells, lower concentrations of carvacrol are needed to obtain the same reduction in viable count of adapted cells. Adapted cells were found to have a lower membrane fluidity, caused by a change in the fatty acid composition and head group composition of the phospholipids in the cytoplasmic membrane. Adaptation to 0.4 mM carvacrol increases the phase transition temperature of the lipid bilayer (T m ) from 20.5°C to 28.3°C. Addition of carvacrol to cell suspensions of adapted B. cereus cells decreases T m again to 19.5°C, approximately the same value as was found for non-adapted cells in the absence of carvacrol.

    Incubation of cooked rice in the presence of different carvacrol concentrations results in a dose-related reduction of the viable count of B. cereus . Concentrations of 0.15 mg/g and above, reduce the viable count, leading to full suppression of growth at 0.38 mg/g. The influence of carvacrol on the viable count is dependent on the initial inoculum size. Although carvacrol is an effective inhibitor of growth of B. cereus in rice, it could affect the flavour and taste of the product at concentrations where full suppression of growth is observed. However, strong synergistic activity is observed when carvacrol is combined with the biosynthetic precursor cymene or the flavour enhancer soya sauce. This makes it possible to use lower carvacrol concentrations and consequently a smaller influence on the sensoric properties of the rice is expected.

    Besides its influence on the viability of vegetative cells, carvacrol also shows inhibition of diarrhoeal toxin production by B. cereus at concentrations below the MIC-value. Addition of 0.06 mg/ml carvacrol to the growth medium, inhibits the toxin to 21% of the control (no carvacrol added). The inhibition correlates with the reduction of the viable count of B. cereus in the presence of carvacrol. At the same time, the total amount of cells did not change. In mushroom soup, also an inhibition of the toxin production was observed, however, the viable count did not change. This effect on the toxin production is most probably caused by a lack of sufficient metabolic energy, since carvacrol affects ATP synthesis. The cell will use its low levels of ATP to maintain its viability, rather than using it for toxin production or excretion. It could also be possible that the decreased toxin synthesis in BHI was the result of the lower amount of viable cells. The inhibition of toxin production at carvacrol concentrations which do permit growth of B. cereus , reduces the risk of food intoxication by this pathogen.

    In conclusion, carvacrol may play an important role in future as a natural antimicrobial compound. However, its application will most probably be in combination with other natural antimicrobial systems.

    Production and excretion of secondary metabolites by plant cell cultures of Tagetes
    Buitelaar, R.M. - \ 1991
    Agricultural University. Promotor(en): J. Tramper. - S.l. : Buitelaar - 159
    asteraceae - weefselkweek - celkweek - meristemen - biochemie - metabolisme - secundaire metabolieten - tagetes - asteraceae - tissue culture - cell culture - meristems - biochemistry - metabolism - secondary metabolites - tagetes

    In this thesis, the results are presented of several approaches to improve the production and excretion of thiophenes by cell cultures or hairy roots of Tagetes spp.

    In chapter one, most of the techniques to improve the production and/or excretion of secondary metabolites with plant cell cultures are discussed in a literature survey. From this chapter, it becomes clear that to date no general rules exist that apply for all plant species. The methods described in this chapter are the introduction of transformed cells in the form of hairy roots, the use of elicitors, the immobilization of plant cells, the use of two-phase cultures and the use of selected fermenters; all have as aim to improve the growth, production and excretion conditions for the plant cell cultures. All these methods have also been tested in the work described in this thesis, with the aim to obtain a higher production of thiophenes for less costs (meaning faster growth and better excretion).

    Chapter two is the description of a side-step towards the extension of an immobilization technique for large scale operation that was introduced a few years before, the resonance nozzle technique. Until then, it had only been tested with alginate, but in this chapter, K -carrageenan, agar and gellan gum were tested as well. The gel formation with these materials is improved when the drops are caught in an organic phase, so the reactions of four cell types (bacteria, yeast, plant cells and insect cells) to several organic solvents and the immobilization were examined. The various cell types appeared to react differently to the applied conditions, but in general hydrophobic solvents proved to be more suitable than hydrophilic solvents.

    The latter phenomenon was elaborated in more detail in the third chapter. Cells of Tagetes minuta , in free or immobilized state, were grown in the presence of various organic solvents and their respiration activity was followed. The introduction of organic solvents can have advantageous effects, like the selective removal of the product from the cells or the aqueous phase, thereby combining production and downstream processing, while the removal of the product can relieve product inhibition effects. There appeared to be a relation between the hydrophobicity of the solvent and the residual activity of the cells - the more hydrophobic the solvent was, the more active the cells were in its presence. The immobilization of the cells provided a slight protection against harmful solvents. Although these Tagetes minuta cells were easy to work with (a fast growing, fine cell suspension), we changed to hairy root cultures of Tagetes patula , transformed with Agrobacterium rhizogenes LBA 9402, because those cells did produce thiophenes, in contrast to the former. These hairy root cultures are more difficult to work with, because they grow as one clump of interconnected roots, so circulation is difficult and samples cannot be taken. Therefore, in chapter four methods are compared that determine the hairy root biomass present in a fermenter in an indirect way. From the three methods extensively tested, i.e. volume/weight balance, oxygen consumption and conductivity measurement, the latter proved to be the simplest and easiest to handle when the roots were in good condition.

    In chapter five, the sensitivity of the hairy roots to organic solvents is described. The results were comparable to those obtained with the Tagetes minuta cell suspensions. An interesting phenomenon was observed with respect to the excretion of thiophenes. In one-phase cultures with only aqueous medium, the excretion is always practically zero. In the presence of some of the solvents tested, e.g. hexadecane, part of the thiophenes produced were excreted into the solvent. When experiments were performed with these two-phase cultures in bioreactors with volumes up to 6 litres, the excretion could rise to about 70% of the total thiophenes produced. Obviously, this is an important result, because it can facilitate the downstream processing of the thiophenes to a great extent. Of the two types of bioreactors tested, the stirred tank reactor and the bubble column, the latter gave the best results for this system.

    In chapter six, similar experiments are described in aqueous two-phase systems. Again, the hairy roots were able to grow and to produce in the aqueous two-phase systems tested, i.e. in shake flasks and bioreactors. In this case, the excretion was stimulated up to 10% of the total thiophene production. In chapter seven, the results are presented of elicitation studies with the Tagetes patula hairy root cultures. The biotic elicitors applied were bacteria, yeast and fungi, while abiotic elicitors were used in the form of extreme pH, Ca 2+addition and the addition of Ethephon, a precursor of ethene. Nearly all elicitor treatments resulted in a higher thiophene production. The highest increase was obtained when the pH was adjusted to 12 (280% of the control production), but the cells were no longer viable after this treatment. The elicitation with extracts of Aspergillus niger led to a production that raised up to 185% of the control, without loss of viability.

    In chapter eight, the combined effects of elicitor treatment and of the use of a solid second phase, XAD-7, is described. Here, the excretion was affected by the addition of the resin, while growth and total production were not influenced, either in the presence or the absence of elicitor and/or XAD.
    Finally, in the general discussion, the obtained results are shortly evaluated.

    Bepaling van glucosinolaat (isothiocyanaat (ITC) + vinylthio-oxazolidon (VTO)
    Essers, M.L. ; Wolters, T.H.C. ; Frijns, L.M.H. ; Muuse, B.G. - \ 1983
    Wageningen : RIKILT (Verslag / RIKILT 83.48) - 16
    analytische methoden - laboratoriummethoden - glycosiden - secundaire metabolieten - oliën - vetten - oliezaden - koolzaad - vloeistofchromatografie - gas-vloeistofchromatografie - hplc - analytical methods - laboratory methods - glycosides - secondary metabolites - oils - fats - oilseeds - rape - liquid chromatography - gas liquid chromatography
    Doel van dit onderzoek: het verkrijgen van een bepalingsmethode voor glucosinolaat in dubbelnul variëteiten van kool/raapzaad (laag glucosinolaat en erucazuurgehalte). Tevens kan deze methode dienen als bepalingsmethode van glucosinolaat in kool/raapschroot t.b.v. denaturatie regeling van melkpoeder. Het glucosinolaatgehalte kan berekend worden uit het ITC en VTO gehalte. Voor het bepalen hiervan bestaat een officieel ISO voorschrift (G.L.C. en spectrofotometrisch). Tevens is er een HPLC-methode ter bepaling van VTO. De genoemde methoden werden getoetst op de mogelijkheid om glucosinolaat t.b.v. dubbelnul variëteiten en voor de denaturatie van melkpoeders te bepalen. Verder werd onderzocht of de ITC en VTO methoden gecombineerd konden worden en of glucosinolaat via het glucose gehalte bepaald kan worden.
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