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.

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    Fungicide-driven evolution and molecular basis of multidrug resistance in field populations of the grey mould fungus Botrytis cinerea
    Kretschmer, M. ; Leroch, M. ; Mosbach, A. ; Walker, A.S. ; Fillinger, S. ; Mernke, D. ; Schoonbeek, H.J. ; Pradier, J.M. ; Leroux, P. ; Waard, M.A. de; Hahn, M. - \ 2009
    PLoS Pathogens 5 (2009)12. - ISSN 1553-7366 - 13 p.
    abc transporter bcatrb - natural toxic compounds - botryotinia-fuckeliana - penicillium-digitatum - candida-albicans - functional-characterization - virulence factor - drug-resistance - gene - sensitivity
    The grey mould fungus Botrytis cinerea causes losses of commercially important fruits, vegetables and ornamentals worldwide. Fungicide treatments are effective for disease control, but bear the risk of resistance development. The major resistance mechanism in fungi is target protein modification resulting in reduced drug binding. Multiple drug resistance (MDR) caused by increased efflux activity is common in human pathogenic microbes, but rarely described for plant pathogens. Annual monitoring for fungicide resistance in field isolates from fungicide-treated vineyards in France and Germany revealed a rapidly increasing appearance of B. cinerea field populations with three distinct MDR phenotypes. All MDR strains showed increased fungicide efflux activity and overexpression of efflux transporter genes. Similar to clinical MDR isolates of Candida yeasts that are due to transcription factor mutations, all MDR1 strains were shown to harbor activating mutations in a transcription factor (Mrr1) that controls the gene encoding ABC transporter AtrB. MDR2 strains had undergone a unique rearrangement in the promoter region of the major facilitator superfamily transporter gene mfsM2, induced by insertion of a retrotransposon-derived sequence. MDR2 strains carrying the same rearranged mfsM2 allele have probably migrated from French to German wine-growing regions. The roles of atrB, mrr1 and mfsM2 were proven by the phenotypes of knock-out and overexpression mutants. As confirmed by sexual crosses, combinations of mrr1 and mfsM2 mutations lead to MDR3 strains with higher broad-spectrum resistance. An MDR3 strain was shown in field experiments to be selected against sensitive strains by fungicide treatments. Our data document for the first time the rising prevalence, spread and molecular basis of MDR populations in a major plant pathogen in agricultural environments. These populations will increase the risk of grey mould rot and hamper the effectiveness of current strategies for fungicide resistance management.
    Overexpression of efflux transporters leads to multidrug resistance in Botrytis cinerea field strains
    Kretschmer, M. ; Walker, A.S. ; Leroch, M. ; Wiwiorra, M. ; Schoonbeek, H. ; Leroux, P. ; Fillinger, S. ; Waard, M. de; Hahn, M. - \ 2008
    In: Book of Abstracts 9th International Congress of Plant Pathology, Torino, Italy, 24-29 August 2008. - - p. S2.140 - S2.140.
    Cloning and functional characterization of BcatrA, a gene encoding an ABC transporter of the plant pathogenic fungus Botryotinia fuckeliana (Botrytis cinerea)
    Sorbo, G. Del; Ruocco, M. ; Schoonbeek, H. ; Scala, F. ; Pane, C. ; Vinale, F. ; Waard, M.A. de - \ 2008
    Mycological Research 112 (2008)6. - ISSN 0953-7562 - p. 737 - 746.
    binding-cassette transporter - natural toxic compounds - saccharomyces-cerevisiae - multidrug-resistance - mycosphaerella-graminicola - drosophila-melanogaster - aspergillus-nidulans - efflux pump - cutinase-a - yeast
    BcatrA was cloned from the plant pathogenic fungus Botryotinia fuckeliana (Botrytis cinerea) and sequenced. Sequence analysis revealed that BcatrA encodes a protein composed of 1562 amino acid residues displaying high similarity with various fungal ATP-binding cassette (ABC) transporters having the (NBF-TM6)2 topology. Expression of BcatrA is barely detectable during normal vegetative growth in liquid substrates. Transcript levels of BcatrA are enhanced in a dose- and time-dependent manner after treatment with cycloheximide or catechol, but not by a number of other drugs or fungicides, including fludioxonil, fenarimol, imazalil, and the plant defense compounds pisatin and resveratrol. Quantitative analysis of BcatrA during the synchronized infection of bean leaves revealed an overaccumulation of the gene transcript at 6, 12 and 24 h post-inoculation, suggesting an involvement of the gene in the first steps of pathogenesis. Functional analysis of BcatrA was performed by targeted gene replacement in a wild-type strain of the fungus, and by overexpression in a mutant of Saccharomyces cerevisiae carrying multiple non-functional multidrug-resistance genes. BcatrA replacement mutants did not show any significant increase in sensitivity to drugs, including inducers of BcatrA transcription, and displayed an unaltered virulence on several common host plants of B. cinerea. However, when expressed in the heterologous system, BcatrA reduced sensitivity to cycloheximide and catechol, thus indicating the ability of the BcatrA product to function as a multidrug transporter.
    Developments in indirect disease control of plant pathogens
    Waard, M. de; Huser, A. ; Roohparvar, R. ; Schoonbeek, H. ; Zwiers, L.H. - \ 2007
    In: Book of Abstracts 14th International Botrytis Symposium, Cape Town, South Africa, 21-26 October 2007. - - p. 62 - 62.
    Impact of fungal drug transporters on fungicide sensitivity, multidrug resistance and virulence
    Waard, M.A. de; Andrade, A.C. ; Hayashi, K. ; Schoonbeek, H. ; Stergiopoulos, I. ; Zwiers, L.H. - \ 2006
    Pest Management Science 62 (2006)3. - ISSN 1526-498X - p. 195 - 207.
    atp-binding cassette - pathogen mycosphaerella-graminicola - azole antifungal agents - natural toxic compounds - yeast abc proteins - candida-albicans - botrytis-cinerea - aspergillus-nidulans - penicillium-digitatum - venturia-inaequalis
    Drug transporters are membrane proteins that provide protection for organisms against natural toxic products and fungicides. In plant pathogens, drug transporters function in baseline sensitivity to fungicides, multidrug resistance (MDR) and virulence on host plants. This paper describes drug transporters of the filamentous fungi Aspergillus nidulans (Eidam) Winter, Botrytis cinerea Pers and Mycosphaerella graminicola (Fu¿ckel) Schroter that function in fungicide sensitivity and resistance. The fungi possess ATP-binding cassette (ABC) drug transporters that mediate MDR to fungicides in laboratory mutants. Similar mutants are not pronounced in field resistance to most classes of fungicide but may play a role in resistance to azoles. MDR may also explain historical cases of resistance to aromatic hydrocarbon fungicides and dodine. In clinical situations, MDR development in Candida albicans (Robin) Berkhout mediated by ABC transporters in patients suffering from candidiasis is common after prolonged treatment with azoles. Factors that can explain this striking difference between agricultural and clinical situations are discussed. Attention is also paid to the risk of MDR development in plant pathogens in the future. Finally, the paper describes the impact of fungal drug transporters on drug discovery
    ABC transporters van Botrytis cinerea in biotische en abiotische interacties
    Schoonbeek, H. - \ 2005
    Gewasbescherming 36 (2005)1. - ISSN 0166-6495 - p. 11 - 13.
    botrytis cinerea - genen - fungicidentolerantie - resistentiemechanismen - genexpressie - botrytis cinerea - genes - fungicide tolerance - resistance mechanisms - gene expression
    Op 29 november 2004 promoveerde Henk-jan Schoonbeek aan Wageningen Universiteit op het proefschrift getiteld 'ABC transporters from Botrytis cinerea in biotic and abiotic interactions'. Promotor was Prof. dr. ir. P.J.G.M. de Wit en co-promotor was M.A. de Waard, leerstoelgroep Fytopathologie, Wageningen Universiteit
    ABC transporters from Botrytis cinerea in biotic and abiotic interactions
    Schoonbeek, H. - \ 2004
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): M.A. de Waard. - [S.l.] : S.n. - ISBN 9789085041085 - 192
    botrytis cinerea - pathogenesis-gerelateerde eiwitten - genen - actief transport - gastheer parasiet relaties - eugenol - genexpressie - plantenziekteverwekkende schimmels - botrytis cinerea - plant pathogenic fungi - pathogenesis-related proteins - genes - gene expression - active transport - host parasite relationships - eugenol
    Botrytis cinereais the causal agent of grey mould disease on a wide variety of crop plants. It is relatively insensitive to natural and synthetic fungitoxic compounds. This thesis describes how ABC (ATP-binding cassette) transporters contribute to protection by actively secreting toxicants from fungal cells, acting as a firsts aid kit. The expression of twelve ABC transporters and three MFS transporters are differentially induced by plant defence compounds, antibiotics and fungicides. BcatrD can decrease sensitivity to azole fungicides, whereas BcatrB is a true multidrug transporter and can provide protection against the plant defence compounds resveratrol and eugenol, phenylpyrrole fungicides and phenazines antibiotics. By exporting resveratrol BcatrB contributes to virulence ofB. cinereaon grapevine. With their ability to protect against fungicides the ABC transporters BcatrB and BcatrD can contribute to the apparent ease by whichB. cinereadevelops resistance against fungicides. The protection against antibiotics is a clear advantage in competition with other microorganisms.
    Drug transporter proteins from Botrytis cinerea function in protection against fungitoxic compounds and biotic interactions
    Waard, M.A. de; Hayashi, K. ; Raaijmakers, J.M. ; Schoonbeek, H. - \ 2004
    In: Book of Abstracts XIII International Botrytis Symposium, Antalya, Turkey, 25-31 October 2004 - p. 113 - 113.
    Pathogen defense against biological control
    Raaijmakers, J.M. ; Schoonbeek, H. ; Schouten, A. ; Waard, M.A. de - \ 2004
    In: Multitrophic interactions in soil and integrated control / Sikora, R.A., Gowen, S., Hauschild, R., Kiewnick, S., Dijon Cedex, France : INRA - Centre de Recherches de Dijon (IOBC wprs bulletin vol. 27(1)) - p. 241 - 245.
    Laccase and ABC-transporters confer resistance in Botrytis cinerea to antibiotics produced by antagonistic Pseudomonas
    Schouten, A. ; Berg-Velthuis, G.C.M. van den; Arenas, Y.C. ; Schoonbeek, H. ; Waard, M.A. de; Raaijmakers, J.M. - \ 2003
    In: Book of Abstracts 6th Plant Growth-Promoting Rhizobacteria Congress, Calicut, India, 5-10 October 2003 [S.l.] : S.n. - p. 585 - 585.
    Laccase and ABC-transporters confer resistance in Botrytis cinerea to antibiotics produced by antagonistic Pseudomonas
    Schouten, A. ; Berg-Velthuis, G.C.M. van den; Arenas, Y.C. ; Schoonbeek, H. ; Waard, M.A. de; Raaijmakers, J.M. - \ 2003
    In: Book of Abstracts 1st Federation of European Microbiological Societies, Ljubljana, Slovenia, 29 June - 3 July 2003 [S.l.] : S.n. - p. 81 - 81.
    Functional analysis of ABC transporter genes from Botrytis cinerea identifies BcatrB as a transporter of eugenol
    Schoonbeek, H. ; Nistelrooy, J.G.M. van; Waard, M.A. de - \ 2003
    European Journal of Plant Pathology 109 (2003)9. - ISSN 0929-1873 - p. 1003 - 1011.
    atp-binding cassette - fungus penicillium-digitatum - multidrug-resistance - toxic compounds - efflux pump - sensitivity - fungicides - pathogenicity - accumulation - resveratrol
    The role of multiple ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporter genes from the plant pathogenic fungus Botrytis cinerea in protection against natural fungitoxic compounds was studied by expression analysis and phenotyping of gene-replacement mutants. The expression of 11 ABC (BcatrA-BcatrK) and three MFS genes (Bcmfs1, Bcmfs2 and Bcmfs4) was studied. All genes showed a low basal level of expression, but were differentially induced by treatment with cycloheximide and the plant defence compounds camptothecin, eugenol, psoralen, resveratrol and rishitin. The latter compounds induced expression of BcatrB at a high level. Eugenol was more toxic to BcatrB gene-replacement mutants than to the control isolates. Eugenol also caused an instantaneous increase in mycelial accumulation of the fungicide fludioxonil, a known substrate of BcatrB. However, there was no difference in virulence between the wild-type and BcatrB gene-replacement mutants on Ocimum basilicum, a plant known to contain eugenol. The results indicate that BcatrB is a transporter of lipophilic compounds, such as eugenol, but its role in virulence remains uncertain.
    Modulators of membrane drug transporters potentiate the activity of the DMI fungicide oxpoconazole against Botrytis cinerea
    Hayashi, K. ; Schoonbeek, H. ; Waard, M.A. de - \ 2003
    Pest Management Science 59 (2003)3. - ISSN 1526-498X - p. 294 - 302.
    sterol demethylation inhibitors - natural toxic compounds - multidrug-resistance - efflux pump - wild-type - botryotinia-fuckeliana - penicillium-italicum - candida-albicans - accumulation - fenarimol
    Modulators known to reduce multidrug resistance in tumour cells were tested for their potency to synergize the fungitoxic activity of the fungicide oxpoconazole, a sterol demethylation inhibitor (DMI), against Botrytis cinerea Pers. Chlorpromazine, a phenothiazine compound known as a calmodulin antagonist, appeared the most potent compound. Tacrolimus, a macrolide compound with immunosuppressive activity, was also active. The synergism of chlorpromazine negatively correlated with the sensitivity of the parent strain and mutants of B cinerea. The synergism was highest in a mutant that overexpressed the ATP-binding cassette transporter BcatrD, known to transport DMI fungicides such as oxpoconazole. The synergism of chlorpromazine positively correlated with its potency to enhance the accumulation of oxpoconazole in BcatrD mutants. These results indicate that chlorpromazine is a modulator of BcatrD activity in B cinerea and suggest that mixtures of DMI fungicides with modulators may represent a perspective for the development of new resistance management strategies.
    Fungal ABC transporters and microbial interactions in natural environments
    Schoonbeek, H. ; Raaijmakers, J.M. ; Waard, M.A. De - \ 2002
    Molecular Plant-Microbe Interactions 15 (2002)11. - ISSN 0894-0282 - p. 1165 - 1172.
    Expression of the ABC transporter BcatrD from Botrytis cinerea reduces sensitivity to sterol demethylation inhibitor fungicides
    Hayashi, K. ; Schoonbeek, H. ; Waard, M.A. De - \ 2002
    Pesticide Biochemistry and Physiology 73 (2002). - ISSN 0048-3575 - p. 110 - 121.
    The ATP-binding cassette (ABC) transporter gene BcatrD from Botrytis cinerea was cloned and characterized. The open reading frame of BcatrD contains seven introns and encodes a putative protein of 1502 amino acids. The function of BcatrD was analyzed by phenotypic characterization of gene-replacement and overexpression mutants. Replacement mutants of BcatrD displayed a higher sensitivity to sterol demethylation inhibitor (DMI) fungicides as compared to the parental isolate. Gene replacement mutants also showed a relatively high accumulation of the DMI fungicide oxpoconazole. Overexpression mutants showed increased levels of basal and oxpoconazole-induced expression of BcatrD. Mutants with the highest expression level displayed the highest decrease in sensitivity to oxpoconazole and a relatively low accumulation of the compound. These results indicate a relation between oxpoconazole sensitivity, expression of BcatrD, and accumulation of oxpoconazole and demonstrate that the ABC transporter BcatrD is a determinant of the sensitivity of B. cinerea to DMI fungicides.
    Bcmfs1, a novel major facilitator superfamily transporter from Botrytis cinerea, provides tolerance towards the natural toxic compounds camptothecin and cercosporin and towards fungicides
    Hayashi, K. ; Schoonbeek, H. ; Waard, M.A. De - \ 2002
    Applied and Environmental Microbiology 68 (2002)10. - ISSN 0099-2240 - p. 4996 - 5004.
    Bcmfs1, a novel major facilitator superfamily gene from Botrytis cinerea, was cloned, and replacement and overexpression mutants were constructed to study its function. Replacement mutants showed increased sensitivity to the natural toxic compounds camptothecin and cercosporin, produced by the plant Camptotheca acuminata and the plant pathogenic fungus Cercospora kikuchii, respectively. Overexpression mutants displayed decreased sensitivity to these compounds and to structurally unrelated fungicides, such as sterol demethylation inhibitors (DMIs). A double-replacement mutant of Bcmfs1 and the ATP-binding cassette (ABC) transporter gene BcatrD was more sensitive to DMI fungicides than a single-replacement mutant of BcatrD, known to encode an important ABC transporter of DMIs. The sensitivity of the wild-type strain and mutants to DMI fungicides correlated with Bcmfs1 expression levels and with the initial accumulation of oxpoconazole by germlings of these isolates. The results indicate that Bcmfs1 is a major facilitator superfamily multidrug transporter involved in protection against natural toxins and fungicides and has a substrate specificity that overlaps with the ABC transporter BcatrD. Bcmfs1 may be involved in protection of B. cinerea against plant defense compounds during the pathogenic phase of growth on host plants and against fungitoxic antimicrobial metabolites during its saprophytic phase of growth
    ATP-binding cassette (ABC) transporters from Botrytis cinerea
    Waard, M.A. De; Hayashi, K. ; Schoonbeek, H. ; Vermeulen, T. - \ 2001
    In: Book of Abstracts XXI Fungal Genetics Conference, Asilomar, USA, 13-18 March 2001 Asilomar, USA : - p. 97 - 97.
    The ABC transporter BcatrB from Botrytis cinerea is a determinant of the activity of the phenylpyrrole fungicide fludioxonil
    Vermeulen, T. ; Schoonbeek, H. ; Waard, M.A. De - \ 2001
    Pest Management Science 57 (2001). - ISSN 1526-498X - p. 393 - 402.
    Multidrug resistance in Botrytis cinerea associated with decreased accumulation of the azole fungicide oxpoconazole and increased transcription of the ABC transporter gene BcatrD
    Hayashi, K. ; Schoonbeek, H. ; Sugiura, H. ; Waard, M.A. De - \ 2001
    Pesticide Biochemistry and Physiology 70 (2001). - ISSN 0048-3575 - p. 168 - 179.
    Azole-resistant mutants of Botrytis cinerea have a multidrug resistance phenotype since they exhibit cross-resistance to unrelated chemicals. These mutants also display resistance to the new azole fungicide oxpoconazole. Resistance to oxpoconazole is associated with decreased accumulation of the fungicide, which is the result of energy-dependent efflux mediated by fungicide transporters. The ATP-binding cassette (ABC) transporter BcatrB (B. cinerea ABC transporter B), involved in efflux of phenylpyrrole fungicides, has no major role in efflux of oxpoconazole since accumulation of the fungicide by a replacement mutant of BcatrB showed a transient accumulation pattern similar to that of the wild-type isolate. The putative role of 10 additional ABC and 3 Major facilitator superfamily transporters in efflux of oxpoconazole was investigated by expression analysis of the corresponding genes. The basal transcription level of BcatrD in germlings of B. cinerea was correlated with the resistance level of two azole-resistant mutants. A short treatment of germlings with the azole fungicides oxpoconazole, prochloraz, and tebuconazole enhanced transcript levels of BcatrD in a wild-type isolate. Transcript levels induced by these fungicides in azole-resistant mutants also correlated with resistance levels. We propose that BcatrD is the ABC transporter that plays a role in azole sensitivity and azole resistance of B. cinerea. Expression of BcatrD is also induced by treatment of germlings with the dicarboximide fungicide iprodione, the benzimidazole fungicide carbendazim, and the antibiotic cycloheximide, suggesting that this gene indeed plays a role in multidrug resistance to fungicides.
    The ABC transporter BcatrB affects the sensitivity of Botrytis cinerea to the phytoalexin resveratrol and the fungicide fenpiclonil
    Schoonbeek, H. ; Sorbo, G. Del; Waard, M.A. De - \ 2001
    Molecular Plant-Microbe Interactions 14 (2001)4. - ISSN 0894-0282 - p. 562 - 571.
    During pathogenesis, fungal pathogens are exposed to a variety of fungitoxic compounds. This may be particularly relevant to Botrytis cinerea, a plant pathogen that has a broad host range and, consequently, is subjected to exposure to many plant defense compounds. In practice, the pathogen is controlled with fungicides belonging to different chemical groups. ATP-binding cassette (ABC) transporters might provide protection against plant defense compounds and fungicides by ATP-driven efflux mechanisms. To test this hypothesis, we cloned BcatrB, an ABC transporter-encoding gene from B. cinerea. This gene encodes a 1,439 amino acid protein with nucleotide binding fold (NBF) and transmembrane (TM) domains in a [NBFTM6]2 topology. The amino acid sequence has 31 to 67% identity with ABC transporters from various fungi. The expression of BcatrB is up regulated by treatment of B. cinerea germlings with the grapevine phytoalexin resveratrol and the fungicide fenpiclonil. BcatrB replacement mutants are not affected in saprophytic growth on different media but are more sensitive to resveratrol and fenpiclonil than the parental isolate. Furthermore, virulence of ΔBcatrB mutants on grapevine leaves was slightly reduced. These results indicate that BcatrB is a determinant in sensitivity of B. cinerea to plant defense compounds and fungicides.
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