Staff Publications

Staff Publications

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

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

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

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

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    Occurrence and diversity of fungal pathogens associated with water hyacinth and their potential as biocontrol agents in the Rift Valley of Ethiopia
    Gebregiorgis, Firehun ; Struik, P.C. ; Lantinga, E.A. ; Tessema, Taye - \ 2017
    International Journal of Pest Management 63 (2017)4. - ISSN 0967-0874 - p. 355 - 363.
    Biological control - Eichhornia crassipes - fungal pathogens - multivariate analysis - native bio-agents - pathogenicity - risk assessment - water hyacinth

    Water hyacinth poses serious socio-economic and environmental problems in Ethiopia. To integrate fungal pathogens into water hyacinth management, a survey was conducted in the Rift Valley of Ethiopia. Based on morphological characterization and DNA sequencing, 25 fungal species were identified that belong to nine genera. Alternaria tenuissima, A. alternata, Aspergillus niger, Phoma sp., Curvularia trifolii, Mucor fragilis, M. racemosus, A. fumigatus, Fusarium oxysporum, and F. equiseti were the most common fungi detected. However, their occurrence was influenced by water wave action, temperature, season, and altitude. Among the fungal pathogens, A. alternata, A. tenuissima, F. oxysporum, F. equiseti, and Neofisicoccum parvum were highly pathogenic to water hyacinth. Alternaria alternata and A. tenuissima did not cause disease symptoms on ecologically important plant species (e.g. Noug, Tef, and Coffee). Application of the fungal pathogens on water hyacinth plants also showed 11%–67%, 22%–72%, 15%–55%, and 12%–50% reduction in fresh weight, dry weight, plant height, and root length of water hyacinth, respectively. This study suggests that fungal species have the potential to control water hyacinth biologically and provides baseline data for biological control efforts in the future.

    Worse comes to worst: bananas and Panama disease—when plant and pathogen clones meet
    Ordonez Roman, N.I. ; Seidl, M.F. ; Waalwijk, C. ; Drenth, A. ; Kilian, A. ; Thomma, B.P.H.J. ; Ploetz, R.C. ; Kema, G.H.J. - \ 2015
    PLoS Pathogens 11 (2015)11. - ISSN 1553-7366 - 7
    bananas - tropical small fruits - agricultural research - fungal diseases - fusarium oxysporum f.sp. cubense - soil fungi - pathogenicity - food production - genetic diversity - plant protection - bananen - tropisch kleinfruit - landbouwkundig onderzoek - schimmelziekten - fusarium oxysporum f.sp. cubense - bodemschimmels - pathogeniteit - voedselproductie - genetische diversiteit - gewasbescherming
    This article deals with: Bananas: their origin and global rollout; genetic diversity of Fusarium oxysporum f.sp. cubense, the causal agent of Panama Disease; Panama Disease: history repeats itself; tropical race 4, a single pathogen clone, threatens global banana production; strategies for sustainable Panama Disease management.
    Transmission of white spot syndrome virus (WSSV) from Dendronereis spp. (Peters) (Nereididae) to penaeid shrimp
    Haryadi, D. ; Verreth, J.A.J. ; Verdegem, M.C.J. ; Vlak, J.M. - \ 2015
    Journal of Fish Diseases 38 (2015). - ISSN 0140-7775 - p. 419 - 428.
    litopenaeus-vannamei - viral accommodation - baculovirus wsbv - scylla-serrata - mud crab - monodon - pathogenicity - host - infection - virulence
    Dendronereis spp. (Peters) (Nereididae) is a common polychaete in shrimp ponds built on intertidal land and is natural food for shrimp in traditionally managed ponds in Indonesia. White spot syndrome virus (WSSV), an important viral pathogen of the shrimp, can replicate in this polychaete (Desrina et al. ); therefore, it is a potential propagative vector for virus transmission. The major aim of this study was to determine whether WSSV can be transmitted from naturally infected Dendronereis spp. to specific pathogen-free (SPF) Pacific white shrimp Litopenaeus vannamei (Boone) through feeding. WSSV was detected in naturally infected Dendronereis spp. and Penaeus monodon Fabricius from a traditional shrimp pond, and the positive animals were used in the current experiment. WSSV-infected Dendronereis spp. and P. monodon in a pond had a point prevalence of 90% and 80%, respectively, as measured by PCR. WSSV was detected in the head, gills, blood and mid-body of Dendronereis spp. WSSV from naturally infected Dendronereis spp was transmitted to SPF L. vannamei and subsequently from this shrimp to new naïve-SPF L. vannamei to cause transient infection. Our findings support the contention that Dendronereis spp, upon feeding, can be a source of WSSV infection of shrimp in ponds.
    Genetic baculovirus determinants for pathogenicity, virulence and transmission
    Serrano, A. - \ 2014
    Wageningen University. Promotor(en): Just Vlak; P. Caballero, co-promotor(en): Gorben Pijlman; D. Munoz. - Wageningen : Wageningen University - ISBN 9789462571358 - 160
    baculovirus - spodoptera exigua multiple nucleopolyhedrovirus - genetische analyse - genotypische variatie - pathogeniteit - virulentie - genen - biologische bestrijding - insectenplagen - baculovirus - spodoptera exigua multiple nucleopolyhedrovirus - genetic analysis - genetic variance - pathogenicity - virulence - genes - biological control - insect pests
    Risk of poultry compartments for transmission of High Pathogenic Avian Influenza
    Boender, G.J. ; Hagenaars, T.H.J. ; Backer, J.A. ; Nodelijk, G. ; Asseldonk, M.A.P.M. van; Bergevoet, R.H.M. ; Roermund, H.J.W. van - \ 2014
    Lelystad : CVI en LEI (Report number CVI: 14/I00028 ) - 28
    aviaire influenza - pluimveeziekten - pluimveehouderij - compartimenten - ziekteoverdracht - pathogeniteit - eu regelingen - wetgeving - diergezondheid - dierenwelzijn - pluimvee - avian influenza - poultry diseases - poultry farming - compartments - disease transmission - pathogenicity - eu regulations - legislation - animal health - animal welfare - poultry
    The application for a poultry compartment by VPI and the prospect of further ones motivates the Dutch Ministry of Economic Affairs and the Netherlands Food and Consumer Product Safety Authority to raise the following three questions: 1) What are the additional transmission risks that the (specific) VPI compartment poses during an HPAI epidemic, compared to a situation without compartment?; 2) What are the additional transmission risks that a compartment in general poses during an HPAI epidemic, depending on its characteristics?; 3) What are relevant evaluation criteria for granting the compartment status? In this study we addressed these questions by quantitatively assessing the veterinary risks based on mathematical model calculations, and by qualitatively discussing the (socio) economic aspects.
    Functional analysis of LysM effectors secreted by fungal plant pathogens
    Kombrink, A. - \ 2014
    Wageningen University. Promotor(en): Bart Thomma; P.J.G.M. de Wit. - Wageningen : Wageningen University - ISBN 9789461738578 - 119
    plantenziekteverwekkende schimmels - secretie - celwanden - chitine - bindende eiwitten - virulentie - pathogeniteit - hyfen - ziekteresistentie - verdedigingsmechanismen - plant pathogenic fungi - secretion - cell walls - chitin - binding proteins - virulence - pathogenicity - hyphae - disease resistance - defence mechanisms

    Chitin is a homopolymer of N-acetyl-d-glucosamine (GlcNAc)that is abundantly present in nature and found as a major structural component in the fungal cell wall. In Chapter 1,the role of chitin as an important factor in the interaction between fungal pathogens and their plant hosts is discussed. As plants do not produce chitin, they evolved to recognize fungal chitin as a non-self molecule by plasma membrane receptors that can activate host immune responses to stop fungal growth.To overcome those host immune responses, fungal pathogens secrete effector molecules that manipulate host physiology, including immune responses, to support colonization. The chitin-binding Lysin motif (LysM) effector Ecp6 from the fungal tomato pathogen Cladosporium fulvumwas previously demonstrated to contribute to virulence through interfering with the activation of chitin-induced host immune responses. Subsequently, LysM effector genes were found in the genomes of many fungal species.

    In Chapter 2 we describe the functional characterization of LysM effectors of the plant pathogenic fungi Mycosphaerella graminicola, Magnaporthe oryzae and Colletotrichum higginsianum, which cause leaf blotch disease of wheat, rice blast disease and anthracnose disease on Brassicaceae, respectively. This functional analysis revealed that the ability to perturb chitin-induced immunity is conserved among LysM effectors of these fungal plant pathogens. In addition, two LysM effectors that are secreted by M. graminicolawere found to protect fungal hyphae against cell wall hydrolytic enzymes from plants, demonstrating that LysM effectors can contribute to virulence of fungal plant pathogens in multiple ways.

    The M. graminicola LysM effector Mg3LysM and C. fulvum Ecp6 both contain three LysM domains and show a high overall similarity. However, whereas Mg3LysM can protect fungal hyphae against plant-derived cell wall hydrolytic enzymes, Ecp6 does not have this capacity. Chapter 3describes a functional analysis of the contribution of LysM domains of Mg3LysM to its protection ability. To this end a series of chimeric proteins were produced in whichLysM domains of Mg3LysM were swapped with the corresponding LysM domain of Ecp6.Analysis of these chimeras indicated that protection against the hydrolytic activity of plant enzymes is mediated by the concerted activity of LysM1 and LysM3 in Mg3LysM.

    LysM effectors do not only occur in foliar fungal plant pathogens, but also in soil-borne pathogens that infect their host through the roots. In Chapter 4, LysM effectors of the fungal soil-borne vascular wilt pathogen Verticillium dahliaeare described. Comparative genomics of eleven V. dahliae strains revealed that four LysM effectors are found in the core genome, which are referred to as core VdLysM effectors. Intriguingly, for none of the core LysM effector genes expression could be monitored during host colonization, and targeted deletion could not reveal a role in virulence, suggesting that the core LysM effectors do not act as virulence factors during host colonization. In addition to the core genome, V. dahliaestrains generally carry lineage-specific (LS) genomic regions. Interestingly, an additional LysM effector gene (Vd2LysM) was found in an LS region of V. dahliaestrain VdLs17 that is absent in all other sequenced V. dahliaestrains. Remarkably, the LS effector Vd2LysM was found to contribute to virulence of strain VdLs17. Like the previously characterized plant pathogen LysM effectors, also Vd2LysM was found to bind chitin and suppress chitin-induced immune responses. These results indicate that Vd2LysM interferes with chitin-induced immunity during host colonization by V. dahliaestrain VdLs17.

    Thus far, LysM effectors were demonstrated to contribute to virulence of various fungal plant pathogens through their ability to interfere with host immune responses. However, the presence of LysM effector genes in the genomes of non-pathogenic fungi and fungi with a saprophytic lifestyle suggests that LysM effectors contribute to fungal physiology in other manners as well. In Chapter 5we investigated the hypothesis that LysM effectors play a role in the interaction of fungi with other microbes in the environment, which could even be relevant for plant pathogenic fungi that encounter other microbes at the site of host infection. To investigate this hypothesis, assays were developed that allow to assess the attachment and antagonistic effects of particular bacterial species on fungi by employing the fungus Trichoderma viride, as this species is known to have accessible cell wall chitin upon growth in vitro. Assays to assess bacterial attachment and antagonistic activity in the absence or presence of LysM effectors indicate that LysM effectors play a role in the protection of fungi against bacterial competitors.

    In Chapter6, the major results described in this thesis are discussed and a perspective on the (potential) roles of LysM effectors in fungi with different lifestyles, including pathogenic as well as non-pathogenic fungi, is presented.

    Insertional mutagenesis in the vascular wilt pathogen Verticillium dahliae
    Santhanam, P. - \ 2014
    Wageningen University. Promotor(en): Bart Thomma, co-promotor(en): Pierre de Wit. - Wageningen : Wageningen University - ISBN 9789461738257 - 124
    plantenziekteverwekkende schimmels - verticillium dahliae - verwelkingsziekten - insertiemutagenese - solanum lycopersicum - tomaten - modellen - pathogeniteit - genkartering - genomica - plant pathogenic fungi - verticillium dahliae - wilts - insertional mutagenesis - solanum lycopersicum - tomatoes - models - pathogenicity - gene mapping - genomics

    Vascular wilt diseases caused by soil-borne pathogens are among the most

    devastating plant diseases worldwide. The ascomycete fungus Verticillium dahliae

    causes vascular wilt diseases in hundreds of dicotyledonous plant species, including

    important crops such as eggplant, lettuce, olive, spinach and tomato. The resting

    structures, microsclerotia, are triggered by root exudates to germinate and penetrate

    the roots after which the fungus grows into the xylem vessels. The fungus colonizes

    these vessels and interferes with the transportation of water and nutrients, resulting in

    the development of symptoms such as stunting, wilting, chlorosis and vascular

    browning. Verticillium wilt diseases are difficult to control due to the longevity of the

    microsclerotia, the broad host range of the pathogen, the inability of fungicides to kill

    the fungus once it has colonized the xylem vessels and the lack of natural resistance in

    many plant species.

    Chapter 1 is the introduction to this thesis that describes the identified

    pathogenicity and virulence factors of V. dahliae and strategies to identify these

    components. In spite of the economic importance of V. dahliae, relatively few

    pathogenicity genes have been identified in this species. With the availability of

    whole genome sequences and the development of functional genomics tools such as

    random mutagenesis, targeted mutagenesis, transcriptomics, RNA interference

    (RNAi) and comparative genomics, more strategies have become available to identify

    novel pathogenicity and virulence genes.

    Chapter 2 focuses on the identification of virulence and pathogenicity genes

    of V. dahliae by screening of a library of random T-DNA insertion mutants. Using

    Agrobacterium tumefaciens-mediated transformation, 900 T-DNA transformants with

    random insertions were generated and screened for altered virulence on susceptible

    tomato plants. This screening, followed by inverse PCR on selected transformants,

    resulted in the identification of 55 potential pathogenicity and virulence genes. One of

    the potential pathogenicity genes, VdNRS/ER, is a homolog of a nucleotide-rhamnose

    synthase/epimerase-reductase (NRS/ER), which is presumably involved in the

    biosynthesis of UDP-rhamnose. Using targeted mutagenesis, VdNRS/ER was deleted

    from wild-type V. dahliae and the resulting deletion mutants were characterized.

    VdNRS/ER deletion mutants exhibit unaltered vegetative growth and sporulation, but

    the deletion mutants were no longer pathogenic on tomato and N. benthamiana and

    showed impaired root attachment on tomato seedlings. These data suggest that UDPrhamnose

    is required for pathogenesis of V. dahliae.

    Chapter 3 describes the role of the V. dahliae homolog of Sge1, a

    transcriptional regulator that was shown to play a role in pathogenicity and regulate

    effector gene expression in Fusarium oxysporum. In this chapter it is demonstrated

    that V. dahliae Sge1 (VdSge1) is required for radial growth and production of asexual

    conidiospores. It is furthermore shown that VdSge1 deletion strains have lost

    pathogenicity on tomato. Since the VdSge1 deletion mutants are not able to infect and

    colonize tomato plants, a tomato cell suspension culture was used to the study the

    expression of Ave1, as well as nine other genes of which the expression is highly

    induced in planta. This assay revealed that VdSge1 is not required for the induction of

    the Ave1 effector that activates resistance mediated by Ve1 in tomato. Furthermore,

    the expression of one other putative effector gene was not affected by VdSge1

    deletion. However, VdSge1 was shown to be required for the expression of six

    putative effector genes, whereas expression of the remaining two putative effectors

    genes was negatively regulated. Thus, the data show that VdSge1 is required for V.

    dahliae pathogenicity and differentially regulates effector gene expression.

    Chapter 4 describes the functional characterization of the gene family

    encoding necrosis- and ethylene-inducing-like proteins (NLPs) of V. dahliae. The

    cytotoxic activity of NLP family members was determined using agroinfiltration into

    tobacco leaves. This resulted in the identification of two out of the seven NLPs,

    VdNLP1 and VdNLP2, that induced plant cell death. The genes encoding these

    cytotoxic NLPs were found to be induced in V. dahliae upon colonization of tomato.

    Targeted deletion of VdNLP1 and VdNLP2 significantly reduced the virulence of V.

    dahliae on tomato and Arabidopsis plants. In contrast, only deletion of VdNLP1

    affected virulence on N. benthamiana whereas deletion of NLP2 did not. However,

    subsequent transcriptional analysis revealed that VdNLP2 was not expressed in V.

    dahliae during colonization of N. benthamiana. Moreover, VdNLP2 also affects

    vegetative growth and conidiospore production. In conclusion, the expanded V.

    dahliae NLP family shows differential cytotoxic activity between family members

    and in planta induction of the cytotoxic NLP genes varies between plant hosts. In

    addition, VdNLP2 plays a role in vegetative growth and conidiospore production in

    addition to its contribution to virulence. Thus, evidence is provided for functional

    diversification within the V. dahliae NLP family.

    Finally in Chapter 5, the major findings of this thesis are discussed and

    placed in a broader perspective.

    Characterisation of Neofusicoccum species causing mango dieback in Italy
    Ismail, A.M. ; Cirvilleri, G. ; Lombard, L. ; Crous, P.W. ; Groenewald, J.Z. ; Polizzi, G. - \ 2013
    Journal of Plant Pathology: rivista di patologia vegetale 95 (2013)3. - ISSN 1125-4653 - p. 549 - 557.
    mangifera-indica - phylogenetic analysis - south-africa - 1st report - sp-nov - botryosphaeria - pathogenicity - lasiodiplodia - morphology - grapevine
    Species of Botryosphaeriaceae are important fungal pathogens of mango worldwide. A survey of 11 mango orchards located in the provinces of Catania, Messina, Palermo and Ragusa (Sicily, southern Italy), resulted in the isolation of a large number (76) of Neofusicoccum isolates associated with decline and dieback symptoms. Isolates were identified based on morphology and DNA sequence data analyses of the internal transcribed spacer region of the nrDNA and partial translation of the elongation factor 1-alpha gene regions. Two species of Neofusicoccum were identified, which included N. parvum and N. australe, the former of which was the dominant species. The high incidence in local orchards and the pathogenicity results indicate that N. parvum and N. australe are important pathogens of mango in Sicily where they may significantly limit mango production.
    Identification and characterization of novel effectors of Cladosporium fulvum
    Ökmen, B. - \ 2013
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Jerome Collemare. - S.l. : s.n. - ISBN 9789461736383 - 190
    solanum lycopersicum - tomaten - plantenziekteverwekkende schimmels - passalora fulva - plant-microbe interacties - verdedigingsmechanismen - genomica - dna-sequencing - pathogeniteit - solanum lycopersicum - tomatoes - plant pathogenic fungi - passalora fulva - plant-microbe interactions - defence mechanisms - genomics - dna sequencing - pathogenicity

    In order to establish disease, plant pathogenic fungi deliver effectors in the apoplastic space surrounding host cells as well as into host cells themselves to manipulate host physiology in favour of their own growth. Cladosporium fulvum is a non-obligate biotrophic fungus causing leaf mould disease of tomato. For decades, this fungus has been a model to study the molecular basis of plant-pathogen interactions involving effector proteins. Characterization of these effectors revealed their roles in both virulence and avirulence as they facilitate colonization of the host in the absence of cognate tomato Cf resistance genes, but also trigger Cf-mediated resistance in the presence of these genes. The availability of the genome sequence of C. fulvum is a great resource allowing us to dissect and better understand the molecular interaction between this fungus and tomato, particularly with regards to identification of new effectors. Such knowledge is of important to improve current strategies not only for disease resistance breeding of tomato against C. fulvum, but also for other host plants that are attacked by pathogenic fungi with similar infection strategies and lifestyles.

    In chapter 1 we give an introduction to the C. fulvum-tomato pathosystem. In a compatible interaction, C. fulvum secretes small cysteine-rich effectors that positively contribute to fungal virulence. Two of these effectors are chitin-binding proteins including Avr4, which protects fungal cell walls against hydrolysis by plant chitinases, and Ecp6, which sequesters released small chitin fragments, thereby preventing induction of basal defense responses associated with their recognition by plant receptors. Another effector, Avr2, is an inhibitor of four tomato cysteine proteases that are also important for basal plant defense. However, in an incompatible interaction, these effectors are directly or indirectly perceived by corresponding resistance proteins (encoded by Cf resistance genes that belong to the class of receptor-like proteins; RLPs) mediating race-specific plant defense responses also known as effector-triggered immunity.

    In chapter 2 we exploit the availability of the genome sequence of C. fulvum to identify novel effectors involved in virulence and avirulence of this fungus. An in silico search was performed using common features of characterized C. fulvum effectors: they (i) contain a signal peptide, (ii) are small (<300 amino acids) and (iii) contain at least four cysteine residues (SSCPs). This search identified 271 SSCPs in the C. fulvum genome. A subset of 60 of these predicted effectors was heterologously expressed in tomato lines carrying different R-traits, including Cf-1, Cf-3, Cf-5, Cf-9B, Cf-11 and Cf-Ecp3 in order to identify the corresponding effectors that are recognized by the RLPs. Although the screen of this subset of SSCPs did not result in identification of a new avirulence gene, two non-specific necrosis-inducing proteins were identified. In addition, a homology search identified CfNLP1, a gene encoding a functional NEP1-like protein that triggers non-specific necrosis in plants. However, quantitative PCR showed that these three genes are lowly or not expressed during tomato infection, which was also true for the in planta expression of some of the effector candidates that were tested for recognition by Cf proteins. In contrast, all genes from C. fulvum encoding the effectors that have been reported so far are highly up-regulated during infection where they play an important role in establishing disease. Like Avr2, Avr4, Ecp2 and Ecp6, we report that Ecp4 and Ecp5 also are involved in virulence of C. fulvum on tomato. Finally, we discuss the limitations of only using bioinformatics approaches to identify novel effectors involved in virulence.

    Inchapter 3 we describe the identification and characterization of a novel effector secreted by C. fulvum. CfTom1 encodes a functional tomatinase enzyme, which belongs to family 10 of glycoside hydrolases (GH10). Bacterial and fungal pathogens of tomato secrete this enzyme to detoxify the toxic saponin, α-tomatine, into the less toxic compounds tomatidine and lycotetraose. Similarly, CfTom1 is responsible for α-tomatine deoxification by C. fulvum both in vitro and during infection of tomato. Accordingly, ∆cftom1 mutants are more sensitive to α-tomatine because they can no longer detoxify α-tomatine. They are less virulent on tomato plants than wild-type as reflected by a delay in disease symptom development and reduced fungal biomass production. In addition, tomatidine appears to be more toxic to tomato cells than α-tomatine, but it does not suppress plant defense responses as previously suggested in literature. Altogether, our results clearly indicate that CfTom1, the major or possibly only tomatinase enzyme produced by C. fulvum, contributes to full virulence of this fungus on tomato by detoxifying α-tomatine.

    Hardly anything is known about in planta regulation of effector genes. In chapter 4 we describe the functional characterization of CfWor1, a homologue of FoSge1, a conserved transcriptional regulator of effectors in Fusarium oxysporum f. sp. lycopersici. CfWor1 is also homologous to Wor1/Ryp1/Mit1 proteins, which are involved in morphological switches in Candida albicans, Histoplasma capsulatum and Saccharomyces cerevisiae, respectively. In contrast to FoSge1, CfWor1 is unlikely a positive regulator of effector genes because it is weakly expressed during infection of tomato. Compared to wild-type, ∆cfwor1 mutants show strong developmental and morphological defects. ∆cfwor1 mutants do not produce any conidia, but differentiate sclerotium-like structures and secrete an extracellular matrix that covers fungal hyphae.∆cfwor1 mutants are no longer virulent on tomato, likely because of developmental defects. Although constitutive expression of CfWor1 in C. fulvum did not cause any obvious developmental defects, except reduced conidia production, the transformants showed reduced virulence. Quantitative PCR on known effector and secondary metabolism genes in both ∆cfwor1 mutants and constitutive expression transformant revealed that the effect of CfWor1 on the expression of these genes is likely due to developmental defects rather than direct regulation. Complementationof a non-virulent ∆fosge1 mutant of F. oxysporum f. sp. lycopersici with full length CfWor1 or chimera of CfWor1 and FoSge1 restored expression of SIX effector genes, but not virulence, indicating that reduced virulence observed for the ∆fosge1 mutant is not solely due to loss of expression ofthese effector genes. Altogether, our study suggests that CfWor1 is a major regulator of development in C. fulvum which indirectly affects virulence.

    Chapter 5 provides a general discussion of the present work on C. fulvum effectors, with particular emphasis on comparative genomics and transcriptomics approaches to identify novel effectors involved in fungal virulence and avirulence. Our findings are put in a broader perspective including a discussion on how identification of effectors will improve our understanding of molecular interactions between plants and pathogenic fungi and how we can use this knowledge to develop new strategies for sustainable disease resistance breeding.

    Brachypodium distachyon line Bd3-1 resistance is elicited by the barley stripe mosaic virus triple gene block 1 movement protein
    Lee, M.Y. ; Yan, L.J. ; Gorter, F.A. ; Kim, B.Y.T. ; Cui, Y. ; Hu, Y. ; Yuan, C. ; Grindheim, J. ; Ganesan, U. ; Liu, Z.Y. ; Han, C.G. ; Yu, J.L. ; Li, D.W. ; Jackson, A.O. - \ 2012
    Journal of General Virology 93 (2012). - ISSN 0022-1317 - p. 2729 - 2739.
    rna-binding - new-model - pathogenicity - determinants - pathogenesis - domain - grass - cdna
    Barley stripe mosaic virus North Dakota 18 (ND18), Beijing (BJ), Xinjiang (Xi), Type (TY) and CV21 strains are unable to infect the Brachypodium distachyon Bd3-1 inbred line, which harbours a resistance gene designated Bsr1, but the Norwich (NW) strain is virulent on Bd3-1. Analysis of ND18 and NW genomic RNA reassortants and RNA beta mutants demonstrates that two amino acids within the helicase motif of the triple gene block 1 (TGB1) movement protein have major effects on their Bd3-1 phenotypes. Resistance to ND18 correlates with an arginine residue at TGB1 position 390 (R-390) and a threonine at position 392 (T-392), whereas the virulent NW strain contains lysines (K) at both positions. ND18 TGB1 R390K ((ND)TGB1(R390K)) and (ND)TGB1(T392K) single substitutions, and an (ND)TGB1(R390K,T392K) double mutation resulted in systemic infections of Bd3-1. Reciprocal (ND)TGB1 substitutions into (NW)TGB1 ((NW)TGB1(K390R) and (NW)TGB1(K392T)) failed to affect virulence, implying that K-390 and K-392 compensate for each other. In contrast, an (NW)TGB1(K390R,K392T) double mutant exhibited limited vascular movement in Bd3-1, but developed prominent necrotic streaks that spread from secondary leaf veins. This phenotype, combined with the appearance of necrotic spots in certain ND18 mutants, and necrosis and rapid wilting of Bd3-1 plants after BJ strain ((BJ)TGB1(K390,T392)) inoculations, show that Bd3-1 Bsr1 resistance is elicited by the TGB1 protein and suggest that it involves a hypersensitive response.
    Molecular typing of Dutch isolates of Xanthomonas arboricola pathovar pruni isolated from ornamental cherry laurel
    Bergsma-Vlami, M. ; Martin, W. ; Koenraadt, H. ; Teunissen, H. ; Pothier, J.F. ; Duffy, B. ; Doorn, J. van - \ 2012
    Journal of Plant Pathology: rivista di patologia vegetale 94 (2012)Supp.1. - ISSN 1125-4653 - p. S29 - S35.
    pectate lyase secretion - acutatum-sensu-lato - colletotrichum-acutatum - c-gloeosporioides - sp-nov - fruit - diversity - infection - susceptibility - pathogenicity
    Xanthomonas arboricola pv. pruni (Xap) has been found in several cherry laurel (Prunus laurocerasus) nurseries in the Netherlands, causing leaf spot. As no information is available yet about the epidemiology of this quarantine bacterium in cherry laurel, molecular typing of Xap isolates can considerably improve our understanding of pathogen spread between various cherry laurel production systems in different regions of the Netherlands and pathogen relatedness among different disease outbreaks. In this study, the genotypic diversity within a population of 25 Xap isolates isolated from different cherry laurel cultivars grown in different locations in the Netherlands between 2008-2010, was assessed using Multiple-locus variable-number analysis (MLVA). The identity of these Xap isolates was initially determined based on the EPPO standard PM 7/64. Confirmation of the identity of these Xap isolates was additionally achieved with diverse methodologies, including gyrase subunit B (gyrB) sequence typing, BOXand ERIC-PCR, AFLP, and Xap-specific PCR’s: one based on the previously described Pagani primers (2004) (conventional-PCR and its TaqMan-PCR variant) and one based on the recently described Pothier primers (2011c). Based on the results of the MLVA analysis, the Dutch population of Xap isolates could be divided into two groups; however no correlation with the geographical origin or any other character of these isolates could be established. Additionally, based on colony morphology, a panel of 5 look-a-likes were isolated from symptomatic leaves of P. laurocerasus which reacted in the Xap-specific PCR described by Pagani (2004) but that did not react in the Xap-specific PCR described by Pothier et al. (2011c). Further characterisation of these look-a-like isolates with AFLP, BOXand ERIC-PCR, and gyrB sequencing showed that the Xap-specific PCR described by Pagani does not discriminate between Xap and the look-a-like isolates. Similarly to Pagani PCR, the performance of a pathogenicity test with a pure culture of the isolate was not always discriminative between Xap and the look-a-like isolates, unraveling a complexity in Xanthomonas pathogenicity. Therefore, in routine screening based on the EPPO standard PM 7/64, complementary techniques such as BOX- ERIC-PCR, gyrB sequencing, Xap-specific PCR described by Pothier (2011c), MLVA and AFLP should be used to obtain a reliable diagnosis of Xap and avoid false positive results.
    Low numbers of repeat units in variable number of tandem repeats (VNTR) regions of white spot syndrome virus are correlated with disease outbreaks
    Tran Thi Tuyet, H. ; Zwart, M.P. ; Phuong, N.T. ; Jong, M.C.M. de; Vlak, J.M. - \ 2012
    Journal of Fish Diseases 35 (2012)11. - ISSN 0140-7775 - p. 817 - 826.
    repetitive dna-sequences - complete genome sequence - ribonucleotide reductase - penaeus-monodon - in-vivo - shrimp - virulence - wssv - identification - pathogenicity
    White spot syndrome virus (WSSV) is the most important pathogen in shrimp farming systems worldwide including the Mekong Delta, Vietnam. The genome of WSSV is characterized by the presence of two major 'indel regions' found at ORF14/15 and ORF23/24 (WSSV-Thailand) and three regions with variable number tandem repeats (VNTR) located in ORF75, ORF94 and ORF125. In the current study, we investigated whether or not the number of repeat units in the VNTRs correlates with virus outbreak status and/or shrimp farming practice. We analysed 662 WSSV samples from individual WSSV-infected Penaeus monodon shrimp from 104 ponds collected from two important shrimp farming regions of the Mekong Delta: Ca Mau and Bac Lieu. Using this large data set and statistical analysis, we found that for ORF94 and ORF125, the mean number of repeat units (RUs) in VNTRs was significantly lower in disease outbreak ponds than in non-outbreak ponds. Although a higher mean RU number was observed in the improved-extensive system than in the rice-shrimp or semi-intensive systems, these differences were not significant. VNTR sequences are thus not only useful markers for studying WSSV genotypes and populations, but specific VNTR variants also correlate with disease outbreaks in shrimp farming systems.
    Stability and variability of virulence of Phytophthorainfestans assessed in a ring test across European laboratories
    Andrivon, D. ; Avendaño-Córcoles, J. ; Cameron, A.M. ; Raaij, H.M.G. van - \ 2011
    Plant Pathology 60 (2011)3. - ISSN 0032-0862 - p. 556 - 565.
    potato-late blight - broad-spectrum resistance - solanum-bulbocastanum - r-gene - dna content - populations - pathogenicity - france - plant - bary
    Determining virulence towards race-specific resistance genes is a prerequisite to understanding the response of pathogen populations to resistant cultivars, and therefore to assess the durability of these resistance genes and the performance of resistance management strategies. In Phytophthora infestans, virulence testing began shortly after the introduction of R-genes from Solanum demissum into S. tuberosum cultivars. However, the characteristics of R-gene expression, the sensitivity of the phenotype to environmental and physiological parameters, and the diversity of experimental protocols make the comparison of data from different studies problematic. This prompted European teams working on P. infestans diversity to: (i) design a joint protocol, using detached leaflets from greenhouse-grown plants of a shared set of differential cultivars inoculated with standardized suspensions of inoculum, and (ii) assess the performance of this protocol in a blind ring test involving 12 laboratories and 10 European isolates of the pathogen. A high level of consensus in the determination of virulence / avirulence to R1, R3, R4, R7, R8, R10 and R11 was achieved among the collaborators, showing that the protocol could be robustly applied across a range of laboratories. However, virulence to R2, R5 or R9 was detected more frequently in some laboratories, essentially from northern Europe; these genes are known to be highly sensitive to host and environmental conditions. The consensus determination was often markedly different from the original virulence phenotype of the isolates, suggesting virulence instability in stored P. infestans isolates. This indicates that creating reliable core collections of pathogen isolates with known virulences could be difficult
    Genome structure and pathogenicity of the fungal wheat pathogen Mycosphaerella graminicola
    M'Barek, S. Ben - \ 2011
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Gert Kema. - [S.l.] : S.n. - ISBN 9789085859970 - 229
    triticum aestivum - triticum turgidum - tarwe - mycosphaerella graminicola - plantenziekteverwekkende schimmels - genoomstructuur - eiwitexpressieanalyse - pathogeniteit - pathogenesis-gerelateerde eiwitten - genomen - plasticiteit - plant-microbe interacties - triticum aestivum - triticum turgidum - wheat - mycosphaerella graminicola - plant pathogenic fungi - genomic structure - proteomics - pathogenicity - pathogenesis-related proteins - genomes - plasticity - plant-microbe interactions

    The phytopathogenic fungus Mycosphaerella graminicola (Fuckel) J. Schröt. in Cohn (asexual stage: Zymoseptoria tritici (Desm.) Quaedvlieg & Crous) causes septoria tritici leaf blotch (STB) in wheat and is one of the most important diseases of this crop worldwide. However, STB control, mainly based on the use of resistant cultivars and fungicides, is significantly hampered by the limited understanding of the genetic and biochemical bases of pathogenicity, and mechanisms of infection and resistance in the host. M. graminicola has a very active sexual cycle under field conditions, which is an important driver of STB epidemics. Moreover, it results in high genetic diversity of field populations that causes a major challenge for the development and sustainable management of resistant cultivars and the discovery of new antifungal compounds. Understanding the role of the sexual and asexual life cycles on genome composition of this versatile pathogen and its infection strategy is crucial in order to develop novel control methods.

    Chapter 1 is an introduction to the biology and pathogenicity of M. graminicola. In addition, it shortly describes the impact of improved and novel technologies on the speed, scope and scale of comparative genomics research.

    Chapter 2 provides detailed genetic analyses of two M. graminicola mapping populations, using mainly DArT markers, and the analysis of the meiotic transmission of unequal chromosome numbers. Polymorphisms in chromosome length and number were frequently observed in progeny isolates, of which 15–20% lacked one or more chromosomes despite their presence in one or both parents, but these had no apparent effect on sexual and pathogenic fitness. M. graminicola has up to eight so called dispensable chromosomes that can be easily lost - collectively called the dispensome - which is, so far, the highest number of dispensable chromosomes reported in filamentous fungi. They represent small-sized chromosomes and make up 38% of the chromosome complement of this pathogen. Much of the observed genome plasticity is generated during meiosis and could explain the high adaptability of M. graminicola in the field. The generated linkage map was crucial for finishing the M. graminicola genome sequence.

    Chapter 3 describes the M. graminicola genome sequence with highlights on genome structure and organization including the eight dispensable chromosomes. The genome comprises a core set of 13 chromosomes and a dispensome, consisting of eight chromosomes that are distinct from the core chromosomes in structure, gene and repeat content. The dispensome contains a higher frequency of transposons and the genes have a different codon use. Most of the genes present one the dispensome are also present on the core chromosomes but little synteny is observed neither between the M. graminicola dispensome and the core chromosomes nor with the chromosomes of other related Dothideomycetes. The dispensome likely originates from ancient horizontal transfer(s) from (an) unknown donor(s).

    Chapter 4 shows a global analysis of proteins secreted by M. graminicola in apoplastic fluids during infection. It focuses mainly on fungal proteins secreted in a compatible interaction. The study showed that many of the annotated secreted proteins have putative functions in fungal pathogenicity, such as cell wall degrading enzymes and proteases, but the function of a substantial number of the identified proteins is unknown. During compatible interactions proteins are primarily secreted during the later stages. However, many pathogenesis-related host proteins, such as PR-2, PR-3 and PR-9, accumulated earlier and at higher concentrations during incompatible interactions, indicating that fungal effectors are recognized by resistant plants and trigger resistant gene-mediated defence responses, though without a visible hypersensitive response.

    Chapter 5 further details the initial identification and characterization of necrosis-inducing proteins that are produced in culture filtrates (CFs) of M. graminicola. The necrosis-inducing activity of CFs is light dependent and inactivated by proteinase K and heat treatment (100C). This is reminiscent of the necrosis-inducing properties of host selective toxins of other Dothideomycete pathogens such as Stagonospora nodorum and Pyrenophora tritici-repentis. Subsequent purifications of CFs and mass spectrometry identified several candidate proteins with necrosis-inducing activity. Heterologous expression of the two most prominent proteins in Pichia pastoris produced sufficient quantities for infiltration assays in a panel of wheat cultivars that showed differential responses, suggesting specific recognition.

    Chapter 6 provides a general discussion of the thesis and puts the results obtained in a broader perspective with a focus on the genome structure of M. graminicola and its function. In addition, aspects of the hemi-biotrophic lifestyle, the relevance of secreted proteins for the wheat-M. graminicola pathosystem in relation to gene-for-gene models and the potential implications for resistance breeding strategies are discussed.

    Eutypa lata, causal agent of dieback in red currant (Ribes rubrum) and gooseberry (R. uva-crispa) in the Netherlands
    Wenneker, M. ; Raak, M. van; Brouwershaven, I.R. van; Martin, W.S. ; Kox, L.F.F. - \ 2011
    European Journal of Plant Pathology 131 (2011)3. - ISSN 0929-1873 - p. 441 - 449.
    1st report - grapevine - identification - pathogenicity - california - cankers - maple
    Dieback of red currant (Ribes rubrum) and gooseberry (Ribes uva-crispa) is an increasing problem in commercial fields in the Netherlands. Field surveys were done in 2006–2007 and samples with dieback symptoms were analysed. In this study the causal agent was diagnosed as Eutypa lata, based on morphological characteristics and rDNA-ITS sequence data. The field surveys revealed the presence of the anamorph and teleomorph states of the fungus produced on dead infected currant wood. Eutypa lata is a vascular pathogen of many woody plants. Related fungi from the same family Diatrypaceae are difficult to distinguish from E. lata based on morphological features. The genetic variability of E. lata was compared by rDNAITS sequencing of isolates from different hosts and origins. Within the E. lata isolates little variability in the ITS sequences was observed. Phylogenetic analysis showed no clear subdivisions within the species. Eutypa lata strains isolated from the different hosts were closely related, indicating that there is no direct evidence for host specificity
    Efficiency of Rz-1 based rhizomania resistance and molecular studies on BNYVV isolates from sugar beet cultivation in Geece
    Pavli, R. ; Prins, M.W. ; Goldbach, R.W. ; Skaracis, G.N. - \ 2011
    European Journal of Plant Pathology 130 (2011). - ISSN 0929-1873 - p. 133 - 142.
    yellow-vein-virus - nucleotide-sequence analysis - united-states - pcr products - rna 5 - identification - pathogenicity - protein - variants - europe
    A survey was carried out to investigate the current situation concerning rhizomania disease incidence in sugar beet cultivation of Greece. A systematic field evaluation over locations and years revealed a consistent disease severity pattern according to favourable agroclimatic conditions and pointed to the so far effectiveness of the Rz1 gene-based resistance, as no major disease outbreaks were observed. Molecular analyses aiming at the characterization of the type and genetic diversity of the virus further confirmed the widespread occurrence of BNYVV in the country, as evidenced by RT-PCR amplification of all five known genomic molecules and nested-PCR assays. None of the isolates contained an RNA 5, typically found in pathotype P. On the basis of RFLP patterns, all BNYVV isolates analysed were classified as pathotype A. Sequence determination of the full-length RNA 3-encoded p25 protein, responsible for symptom development, revealed amino acid motifs ACHG/VCHG in the hypervariable region aa67–70. The presence of valine in position 67 did not appear associated with increased pathogenicity and resistance breaking properties, as earlier reported.
    Development of Metarhizium anisopliae and Beauveria bassiana formulations for control of malaria mosquito larvae
    Bukhari, S.T. ; Takken, W. ; Koenraadt, C.J.M. - \ 2011
    Parasites & Vectors 4 (2011). - ISSN 1756-3305 - 23 p.
    entomopathogenic fungus - aedes-aegypti - culex-quinquefasciatus - anopheles-stephensi - western kenya - gambiae-s.s. - conidia - pathogenicity - vectors - insecticide
    Background The entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana have demonstrated effectiveness against anopheline larvae in the laboratory. However, utilising these fungi for the control of anopheline larvae under field conditions, relies on development of effective means of application as well as reducing their sensitivity to UV radiation, high temperatures and the inevitable contact with water. This study was conducted to develop formulations that facilitate the application of Metarhizium anisopliae and Beauveria bassiana spores for the control of anopheline larvae, and also improve their persistence under field conditions. Methods Laboratory bioassays were conducted to test the ability of aqueous (0.1% Tween 80), dry (organic and inorganic) and oil (mineral and synthetic) formulations to facilitate the spread of fungal spores over the water surface and improve the efficacy of formulated spores against anopheline larvae as well as improve spore survival after application. Field bioassays were then carried out to test the efficacy of the most promising formulation under field conditions in western Kenya. Results When formulated in a synthetic oil (ShellSol T), fungal spores of both Metarhizium anisopliae and Beauveria bassiana were easy to mix and apply to the water surface. This formulation was more effective against anopheline larvae than 0.1% Tween 80, dry powders or mineral oil formulations. ShellSol T also improved the persistence of fungal spores after application to the water. Under field conditions in Kenya, the percentage pupation of An. gambiae was significantly reduced by 39 - 50% by the ShellSol T-formulated Metarhizium anisopliae and Beauveria bassiana spores as compared to the effects of the application of unformulated spores. Conclusions ShellSol T is an effective carrier for fungal spores when targeting anopheline larvae under both laboratory and field conditions. Entomopathogenic fungi formulated with a suitable carrier are a promising tool for control of larval populations of malaria mosquitoes. Additional studies are required to identify the best delivery method (where, when and how) to make use of the entomopathogenic potential of these fungi against anopheline larvae.
    First report of the infection of insecticide-resistant malaria vector mosquitoes with an entomopathogenic fungus under field conditions
    Howard, A.F.V. ; N'Guessan, R. ; Koenraadt, C.J.M. ; Asidi, A. ; Farenhorst, M. ; Akogbeto, M. ; Knols, B.G.J. ; Takken, W. - \ 2011
    Malaria Journal 10 (2011). - ISSN 1475-2875 - 8 p.
    anopheles-gambiae - culex-quinquefasciatus - metarhizium-anisopliae - beauveria-bassiana - conidial thermotolerance - west-africa - efficacy - benin - nets - pathogenicity
    Background - Insecticide-resistant mosquitoes are compromising the ability of current mosquito control tools to control malaria vectors. A proposed new approach for mosquito control is to use entomopathogenic fungi. These fungi have been shown to be lethal to both insecticide-susceptible and insecticide-resistant mosquitoes under laboratory conditions. The goal of this study was to see whether entomopathogenic fungi could be used to infect insecticide-resistant malaria vectors under field conditions, and to see whether the virulence and viability of the fungal conidia decreased after exposure to ambient African field conditions. Methods - This study used the fungus Beauveria bassiana to infect the insecticide-resistant malaria vector Anopheles gambiae s.s (Diptera: Culicidae) VKPER laboratory colony strain. Fungal conidia were applied to polyester netting and kept under West African field conditions for varying periods of time. The virulence of the fungal-treated netting was tested 1, 3 and 5 days after net application by exposing An. gambiae s.s. VKPER mosquitoes in WHO cone bioassays carried out under field conditions. In addition, the viability of B. bassiana conidia was measured after up to 20 days exposure to field conditions. Results - The results show that B. bassiana infection caused significantly increased mortality with the daily risk of dying being increased by 2.5× for the fungus-exposed mosquitoes compared to the control mosquitoes. However, the virulence of the B. bassiana conidia decreased with increasing time spent exposed to the field conditions, the older the treatment on the net, the lower the fungus-induced mortality rate. This is likely to be due to the climate because laboratory trials found no such decline within the same trial time period. Conidial viability also decreased with increasing exposure to the net and natural abiotic environmental conditions. After 20 days field exposure the conidial viability was 30%, but the viability of control conidia not exposed to the net or field conditions was 79%. Conclusions - This work shows promise for the use of B. bassiana fungal conidia against insecticide-resistant mosquitoes in the field, but further work is required to examine the role of environmental conditions on fungal virulence and viability with a view to eventually making the fungal conidia delivery system more able to withstand the ambient African climate.
    Passaging of a Newcastle disease virus pigeon variant in chickens results in selection of viruses with mutations in the polymerase complex enhancing virus replication and virulence
    Dortmans, J.C.F.M. ; Rottier, P.J.M. ; Koch, G. ; Peeters, B.P.H. - \ 2011
    Journal of General Virology 92 (2011)2. - ISSN 0022-1317 - p. 336 - 345.
    avian paramyxovirus type-1 - fusion protein - great-britain - cleavage site - influenza-virus - molecular-basis - rna-polymerase - pmv-1 viruses - pathogenicity - host
    Some Newcastle disease virus (NDV) variants isolated from pigeons (pigeon paramyxovirus type 1; PPMV-1) do not show their full virulence potential for domestic chickens but may become virulent upon spread in these animals. In this study we examined the molecular changes responsible for this gain of virulence by passaging a low-pathogenic PPMV-1 isolate in chickens. Complete genome sequencing of virus obtained after 1, 3 and 5 passages showed the increase in virulence was not accompanied by changes in the fusion protein – a well known virulence determinant of NDV – but by mutations in the L and P replication proteins. The effect of these mutations on virulence was confirmed by means of reverse genetics using an infectious cDNA clone. Acquisition of three amino acid mutations, two in the L protein and one in the P protein, significantly increased virulence as determined by intracerebral pathogenicity index tests in day-old chickens. The mutations enhanced virus replication in vitro and in vivo and increased the plaque size in infected cell culture monolayers. Furthermore, they increased the activity of the viral replication complex as determined by an in vitro minigenome replication assay. Our data demonstrate that PPMV-1 replication in chickens results in mutations in the polymerase complex rather than the viral fusion protein, and that the virulence level of pigeon paramyxoviruses is directly related to the activity of the viral replication complex.
    Two genetically closely related pigeon paramyxovirus type 1 (PPMV-1) variants with identical velogenic fusion protein cleavage sites but with strongly contrasting virulence
    Dortmans, J.C.F.M. ; Fuller, C.M. ; Aldous, E.W. ; Rottier, P.J.M. ; Peeters, B.P.H. - \ 2010
    Veterinary Microbiology 143 (2010)2-4. - ISSN 0378-1135 - p. 139 - 144.
    newcastle-disease virus - hemagglutinin-neuraminidase protein - nucleotide-sequence - monoclonal-antibodies - great-britain - pmv-1 viruses - pathogenicity - passage - isolate - expression
    Two pathogenetically different pigeon paramyxovirus type 1 (PPMV-1) virus clones were recently derived by passage of a single isolate with an intracerebral pathogenicity index (ICPI) of 0.32. The virus clones had an ICPI of 0.025 and 1.3, respectively (Fuller et al., 2007). Remarkably both viruses contained a cleavage site motif in the precursor fusion (F) protein that is usually associated with virulent viruses. In the current study, both viral genomes were completely sequenced and only four amino acid differences were observed. Of these, two were considered irrelevant on theoretical grounds and two amino acid changes were unique for virus 0.025. The latter were introduced into an infectious clone of a virulent Newcastle disease virus strain, individually and combined, and the effects of the mutations on pathogenicity were examined. The results indicate that only the S453P substitution in the F protein had a modest effect on pathogenicity. We were not able to identify the molecular basis for the pathogenicity difference between both viruses. However, our observations emphasize the need to determine both the virulence (ICPI) and the sequence of the cleavage site of the F protein to avoid dismissing of potential virulent PPMV-1 isolates.
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