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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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    Antibiotic resistance reservoirs : the cases of sponge and human gut microbiota
    Versluis, Dennis - \ 2016
    Wageningen University. Promotor(en): Hauke Smidt, co-promotor(en): Mark van Passel; Detmer Sipkema. - Wageningen : Wageningen University - ISBN 9789462579057 - 197
    antibiotic resistance - reservoirs - intestinal microorganisms - luffa - forest soils - sediment - escherichia coli - penicillium - faecal examination - antibioticaresistentie - reservoirs - darmmicro-organismen - luffa - bosgronden - sediment - escherichia coli - penicillium - fecesonderzoek

    One of the major threats to human health in the 21st century is the emergence of pathogenic bacteria that are resistant to multiple antibiotics, thereby limiting treatment options. An important route through which pathogens become resistant is via acquisition of resistance genes from environmental and human-associated bacteria. Yet, it is poorly understood to what extent and by what mechanisms these so-called reservoirs contribute to emerging resistance. Therefore, the work described in this thesis focussed on generating novel insights into different niches as sources of resistance, with a particular focus on the human gut microbiota as well as on microbial communities associated with marine sponges, especially because the latter have been described as one of the richest sources of bioactive secondary metabolites, including a broad range of antimicrobials. Cultivation-based methods were complemented with culture-independent approaches in order to study bacterial taxa that are not readily cultivated.

    Using metatranscriptomics it was found that clinically relevant antibiotic resistance genes are expressed in a broad range of environmental niches including human, mouse and pig gut microbiota, sea bacterioplankton, a marine sponge, forest soil and sub-seafloor sediment. The diversity of resistance gene transcripts differed greatly per niche indicating that the environment contains a rich reservoir of functional resistance that could be accessible by pathogens. Even though resistance gene expression might be linked to the presence of natural antibiotics, we did not detect expression of the corresponding secondary metabolite biosynthesis clusters.

    Thirty-one antibiotic-resistant bacteria, amongst which three belonging to potentially novel Flavobacteriaceae spp., were isolated from the Mediterranean sponges Aplysina aerophoba, Corticium candelabrum and Petrosia ficiformis. Isolates were identified in a high throughput manner by double-barcoded 16S rRNA gene amplicon sequencing. Furthermore, analysis of sponge tissue-derived bacterial biomass growing on agar media showed that many novel bacterial taxa can still be isolated by conventional cultivation methods. Genomic DNA from the 31 antibiotic resistant bacteria was interrogated with respect to the presence of active resistance genes by functional metagenomics. In addition, we also screened metagenomic libraries prepared from DNA directly isolated from sponge tissue in order to circumvent the need for cultivation. In total, 37 unique resistance genes were identified, and the predicted gene products of 15 of these shared <90% amino acid identity with known gene products. One resistance gene (blaPSV-1), which was classified into a new β-lactamase family, was found to be exclusive to the marine specific genus Pseudovibrio. These findings raised questions as to the functional roles of these genes in sponges, but more importantly, the functionality of these genes in E. coli shows that they can potentially be harnessed by phylogenetically distinct bacteria in other environments, including human pathogens. As such, it is a wake-up call as to the significance of marine resistance reservoirs.

    Pseudovibrio, a genus of α-Proteobacteria, was studied in more detail by comparative genomics as it comprises bacteria that potentially play a role as sponge symbionts and marine hubs of antibiotics resistance. Based on gene content, members of the genus Pseudovibrio were found to cluster by sponge sampling location indicating geographic speciation. Furthermore, Pseudovibrio spp. isolated from sponges near the Spanish coast clustered by sponge, suggesting host-specific colonization or adaptation. Strong support for Pseudovibrio spp. forming symbiotic relations with sponges came from the presence of a plethora of (predicted) conserved symbiosis-related functions in their genomes.

    A final study aimed to isolate novel antibiotic resistant reservoir species from the human gut microbiota using a targeted approach. Faecal samples from hospitalized patients that received Selective Digestive Decontamination (SDD), a prophylactic treatment with a cocktail of different antibiotics (tobramycin, polymyxin E, amphotericin B and cefotaxime), were inoculated anaerobically on agar media, after which bacterial biomass was analysed by 16S rRNA gene amplicon sequencing. Six novel taxa were identified that, based on their growth on media supplemented with the SDD antibiotics, could serve as clinically relevant reservoirs of antibiotic resistance. For one of these six taxa a member was obtained in pure culture by targeted isolation. The abundance of antibiotic resistant uncultivated taxa in the human gut microbiota warrants further research as to their potential roles in resistance dissemination.

    In conclusion, this thesis provides deeper insights into different environmental niches as reservoirs of antibiotic resistance. The results can serve to prime and inspire future research.

    Koken en bolontsmetting narcis zonder formaline
    Vreeburg, P.J.M. ; Korsuize, C.A. - \ 2014
    Lisse : Praktijkonderzoek Plant en Omgeving BBF - 19
    narcissus - bloembollen - fusarium - penicillium - desinfectie - alternatieve methoden - ontsmettingsmiddelen - ziektebestrijding - heetwaterbehandeling - narcissus - ornamental bulbs - fusarium - penicillium - disinfection - alternative methods - disinfectants - disease control - hot water treatment
    Bij narcis valt het gebruik van formaline weg als basis voor de bolontsmetting bij voorweken, koken en de ontsmetting vlak voor planten. In dit PPO-onderzoek is een aantal alternatieve middelen(combinaties) getest op hun werking tegen Fusarium bolrot, huidziek en Penicillium en beoordeeld op invloed op de opbrengst. Captan en combinaties van prochloraz + thiofanaat-methyl waren instaat formaline goed te vervangen ten aanzien van de opbrengst en huidkwaliteit. Als de bollen na het koken nog worden verwerkt is het gebruik van captan sterk af te raden i.v.m. blootstellingsrisico’s. De bestrijding van bolrot viel in alle gevallen zwaar tegen. Zowel bij de oude adviezen met formaline als bij de alternatieve middelencombinaties zonder formaline trad zeer veel bolrot op. Achteraf bleek de gebruikte partij Dutch Master zeer zwaar latent besmet te zijn met Fusarium. Een conclusie ten aanzien van de bolrotbestrijding viel daardoor niet met zekerheid te trekken. Vervolgonderzoek is noodzakelijk om na te gaan of formaline voor het voorkomen van de bolrotverspreiding, kan worden vervangen door de andere middelen captan, prochloraz en thiofanaat-methyl. De werking van de middelen werden niet meetbaar beïnvloed door het tijdstip van de warmwaterbehandelingen (vroeg of laat) met of zonder voorweken. Getest is zowel bij voorweken gevolgd door een warmwaterbehandeling van 4 uur 47°C, als bij een vroege (half augustus) en late (half september) warmwaterbehandeling bij 2 uur 45°C. Vroeg koken leidde soms wel tot meer bolrot dan laat koken. De geteste cultivars waren Tête-à-Tête (o.a. Penicillium), Barrett Browning (o.a. huidkwaliteit) en Dutch Master (o.a. bolrot).
    Penicillium in lelie : Effect van terugdrogen na het spoelen op Penicilliumaantasting tijdens de bewaring van lelie
    Kok, B.J. - \ 2010
    Lisse : PPO Bloembollen en Bomen - 13
    penicillium - lilium - lelies - plantmateriaal - opslag - plantenziektebestrijding - bloembollen - penicillium - lilium - lilies - planting stock - storage - plant disease control - ornamental bulbs
    Penicilliumaantasting in leverbare leliebollen tijdens de bewaring in ijs is niet te voorkomen door de bollen na het spoelen na de oogst terug te drogen bij een hoge RV en een hoge temperatuur. De mate van aantasting door Penicillium was na een half jaar bewaring in ijs, van bollen die relatief warm en vochtig zijn gedroogd, niet significant lager dan de mate van aantasting in bollen die bij hoge RV en lagere temperaturen werden teruggedroogd zoals in de praktijk gebeurt. Wederom werd bevestigd dat te onrijp geoogste bollen extreem gevoelig zijn voor Penicillium. Leliebollen kunnen tijdens de bewaring in het ijs door Penicillium worden aangetast. Een leliebol die door Penicillium is aangetast geeft in de broeierij een lichtere tak of kan zelfs uitvallen en helemaal geen tak geven. De laatste jaren komen er steeds meer klachten uit de praktijk (broeierij en export) over Penicillium tijdens de bewaring van lelies. Als mogelijke oorzaak voor de stijging van Penicillium wordt door leliebroeiers vaak verwonding door het gebruik van de aquagrader genoemd. De aquagrader is een mes dat bolwortels inkort tijdens het spoelen. Door een geringere wortelmassa zou er in de verwerking van de bollen meer beschadiging optreden. Na het spoelen worden leliebollen in kuubkisten opgevangen. Door de geringere hoeveelheid wortels zouden de schubben tijdens de verwerking sneller beschadigen. In de praktijk worden de leliebollen na het spoelen snel teruggedroogd, wat ongunstig is voor wondheling. Uit oud onderzoek is bekend dat lelieschubben door snel drogen veel gevoeliger zijn voor Penicillium, doordat de wondheling wordt vertraagd (en Penicilliumsporen de open wond kunnen binnengroeien). Uit onderzoek van E. Roebroeck (LBO) in 1987 is gebleken dat de kans op Penicillium in lelieschubben met 60 tot 95% afneemt door de schubben direct na het schubben een dag juist warm (20°C) en vochtig (95% RV) te bewaren waardoor de wondheling wordt gestimuleerd.
    Voorstudie detectie en risico-inschatting vruchtrot : tussentijdse rapportage
    Heijne, B. - \ 2009
    Zoetermeer : Produktschap Tuinbouw, afdeling Markt & Innovatie - 3
    gewasbescherming - appels - schimmelziekten - fruitteelt - detectie - monilinia - penicillium - gloeosporium - vruchtrot - risicoschatting - plantenziekten - plant protection - apples - fungal diseases - fruit growing - detection - monilinia - penicillium - gloeosporium - fruit rots - risk assessment - plant diseases
    Het doel van dit project is het in kaart brengen welke vruchtrotziekten op welk moment infectiedruk veroorzaken. Voor deze ziekten nagaan welke detectie technieken geschikt zouden kunnen zijn om een risico-inschatting per perceel of boomgaard te maken. Dit wordt gedaan voor appel en peer.
    Methoden ter voorkóming van Penicilliumaantasting tijdens de bewaring van tulpenbollen
    Dam, M.F.N. van - \ 2009
    Lisse : PPO Bloembollen en Bomen - 21
    tulipa - tulpen - penicillium - opslag - plantenziektebestrijding - bloembollen - bollen - tulipa - tulips - penicillium - storage - plant disease control - ornamental bulbs - bulbs
    Tulpen in de droogverkoop moeten er goed uit zien: goed in de huid en geen zichtbare beschadiging of aantastingen door schimmel. Tegenwoordig worden tulpen ook op de bol in glas of in andere verpakking verkocht. Ook daar is uitwendige schimmelgroei snel zichtbaar en kan dit leiden tot afwijzing. Bollen vrij houden van uitwendige schimmelgroei begint met het voorkomen van beschadigingen. Daarna is het zaak de bollen goed droog te bewaren. Dat is niet altijd mogelijk, want tijdens transport en bewaring kan op verschillende momenten de luchtvochtigheid oplopen, vooral tijdens koeling. Bestrijding van Penicillium met gewasbeschermingsmiddelen is voor bollen, bestemd voor de broeierij, ongewenst en voor bollen voor de droogverkoop onacceptabel. Er is daarom onderzoek uitgevoerd naar stoffen en methoden om Penicillium te bestrijden die milieuvriendelijk zijn. In het onderzoek is gewerkt aan het milieuvriendelijke fungicide Delvocid®Instant, etherische oliën (3 soorten) en aan ruimteontsmetting met ioniserende apparatuur AiroCide®. Dit onderzoek heeft opgeleverd dat een dompeling van bollen in het schimmelwerende middel Delvocid®Instant penicilliumgroei op de bollen onder vochtige omstandigheden voorkómt. De preventieve werking van het middel was vooral zichtbaar door het wegblijven van schimmelpluis en -sporen op verwondingen en stootplekjes. Dit effect is des te gunstiger omdat Penicillium juist op die plaatsen makkelijk ontstaat. Met de 3 geteste etherische oliën kon een vermindering van uitwendig Penicillium op de bollen worden gerealiseerd ten opzichte van onbehandelde bollen. Er bleek ook een schimmelreducerend effect te zijn op Penicillium op wondjes. Een bezwaar van deze groep van middelen is dat er bij verkeerd gebruik ook schade door de damp kan ontstaan. Dit uit zich in irritatie aan de wortelkrans en verkleuringen van de bolrok. Verder onderzoek naar de dosering en de toedieningswijze is daarom nog nodig. De AiroCide® luchtontsmetter bleek in dit onderzoek niet effectief in het verminderen van het aantal sporen in de lucht. Het aantal sporen in de luchtstroom van de bewaarcel nam na enkele uren wel af, maar dit werd niet versneld door het gebruik van de ontsmetter. Tijdens deze proef kwam een situatie voor waaruit bleek dat stoffilters waarschijnlijk ook effectief kunnen bijdragen aan de afname van het aantal sporen in de lucht.
    UV tegen ziekten : tussenrapportage - "proof of principle" UV-C tegen schurft, meeldauw en bewaarrot
    Heijne, B. ; Wenneker, M. ; Joosten, N.N. ; Anbergen, R.H.N. - \ 2008
    Randwijk : Wageningen UR, Praktijkonderzoek Plant & Omgeving, Sector Fruit - 76
    schimmelziekten - appels - peren - plantenziekten - botrytis - gloeosporium - penicillium - colletotrichum - monilia - ziektebestrijding - ultraviolette straling - schimmelsporen - mycelium - fungal diseases - apples - pears - plant diseases - botrytis - gloeosporium - penicillium - colletotrichum - monilia - disease control - ultraviolet radiation - fungal spores - mycelium
    Rapportnr.: 2008-30. - Projectnr.: 3261071700. - PT-nr: 13067
    Minderbewaarproblemen door juiste temperatuur tussen oogst en inpakken
    Kok, B.J. ; Gude, H. - \ 2008
    BloembollenVisie 2008 (2008)152. - ISSN 1571-5558 - p. 20 - 21.
    bloembollen - lelies - lilium - opslag - spruiten - penicillium - opslagkwaliteit - bewaarziekten - ornamental bulbs - lilies - storage - sprouts - storage quality - storage disorders
    Tussen oogst en afleveren van lelies is het van groot belang om zorgvuldig om te gaan met de opslag van de bollen. Bollen die te lang warm hebben gestaan lopen een verhoogd risico op zwarte spruiten. PPO-onderzoek heeft aangetoond hoe de kans op zwarte spruiten tot een minimum is te beperken
    Beheersing en detectie van wortelbederf, veroorzaakt door Fusarium-soorten in broeitulpen
    Doorn, J. van; Vink, P. ; Hollinger, T.C. - \ 2005
    Lisse : PPO Bloembollen - 33
    fusarium - trichoderma - botrytis cinerea - penicillium - gibberella avenacea - tulipa - forceren van planten - potcultuur - plantenziektebestrijding - landbouwkundig onderzoek - detectie - tulpen - fusarium - trichoderma - botrytis cinerea - penicillium - gibberella avenacea - tulipa - forcing - pot culture - plant disease control - agricultural research - detection - tulips
    Bij de broeierij van tulpen op potten en bakken met potgrond ontstaat vaak op en onder de bodem een dikke laag wortels. Deze wortels zijn meestal niet omsloten door grond en kunnen onder bepaalde omstandigheden worden aangetast door schimmels als Trichoderma, Botrytis cinerea, Penicillium en Fusarium avenaceum. Enkele jaren geleden was plotseling sprake van veel problemen met een wortelbederf veroorzaakt door een schimmel waardoor de tulpen te kort bleven en op de bladeren grijze bladvlekken ontstonden. Deze symptomen konden niet worden verklaard met de hierboven genoemde schimmels. Isolaties uit aangetaste wortels bracht aan het licht dat het om een Fusarium-schimmel ging, maar in hoeverre deze schimmel nu werkelijk verantwoordelijk was voor de aantasting bleef onduidelijk. Daarom is binnen dit project onderzoek gedaan om na te gaan of de gevonden Fusarium-schimmels verantwoordelijk zijn voor een wortelbederf bij broeitulpen waardoor de tulpen te kort blijven en grijze bladvlekken kunnen ontstaan. Tevens is geprobeerd om aan te tonen dat andere substraten dan potgrond wortelbederf op de bodem van potten en bakken kan beperken. Uit het onderzoek is gebleken dat naast schimmels als Trichoderma en Fusarium avenaceum ook de schimmel Fusarium culmorum een wortelbederf bij tulpen kan veroorzaken. Als gevolg van een aantasting door deze schimmel ontwikkelen de tulpen zich trager en blijven daardoor te kort. Bovendien ontstaan soms symptomen in het blad die sterke verwantschap hebben met dat van Trichoderma-bladtopverdorring. Ook is gebleken uit de infectieproeven dat wortelbederf veroorzaakt door de schimmel Fusarium avenaceum juist langere en slappere tulpen veroorzaakt. Wanneer een ander substraat dan potgrond, zoals zand of perlite op de bodem van potten werd aangebracht kon wortelbederf door Fusarium culmorum iets worden beperkt. De beheersbaarheid van wortelbederf op de bodem van potten en bakken berust voornamelijk op teeltmaatregelen die bekend zijn bij wortelbederf door Trichoderma en Botrytis cinerea. Een artikel over wortelbederf in broeitulpen is verschenen en hierin is een inventarisatie gemaakt, deels berustend op al eerder uitgevoerd onderzoek bij Diagnostiek van PPO, over beheersmaatregelen om wortelbederf in tulp te voorkomen. Dit artikel is later vertaald en ter beschikking gesteld aan buitenlandse broeiers. Er is tevens onderzoek gedaan om zowel Fusarium avenaceum als Fusarium culmorum middels DNA-technieken aan te kunnen tonen. Met de ontwikkelde PCR-toets bleken beide Fusarium-soorten te kunnen worden aangetoond en tevens werd het mogelijk om beide soorten van elkaar te onderscheiden. Praktijkmonsters kunnen nu geanalyseerd worden op aanwezigheid van deze schimmels. Met het beschikbaar komen van een specifieke DNA-toets voor het aantonen van Fusarium avenaceum en Fusarium culmorum werd het ook mogelijk om na te gaan waar en hoe een besmetting in het broeisysteem terecht zou kunnen komen. Daarom is in dit deel van het onderzoek nagegaan of Fusarium culmorum op Nederlandse tulpenbollen, op afdekstro of in grond waarop in Scandinavië de tulpenbollen worden geplant kon worden aangetoond. Op Nederlandse tulpenbollen en in Scandinavische grondmonsters werd geen Fusarium culmorum gevonden. Op Nederlands stro werd deze schimmel wel aangetoond. Dat is ook niet verbazend, want Fusariumculmorum is een bekende pathogeen in granen. Toch is hiermee niet duidelijk geworden via welke route de wortels van tulpen besmet kunnen raken met Fusarium culmorum waardoor wortelbederf kan ontstaan. Tijdens bijeenkomsten met de begeleidingscommissie (leden Scandinavië-groep KBGBB) op 23 oktober 2003 en 5 oktober 2004 is verslag gedaan van de uitgevoerde proeven en resultaten.
    Warm drogen voorkomt Penicillium bij Tete-a-Tete
    Vreeburg, P.J.M. ; Korsuize, C.A. - \ 2005
    BloembollenVisie (2005)65. - ISSN 1571-5558 - p. 22 - 23.
    bloembollen - narcissus - plantenziekteverwekkende schimmels - penicillium - opslag - hoge temperatuurdroging - klimaatregeling - ventilatie - landbouwkundig onderzoek - ornamental bulbs - narcissus - plant pathogenic fungi - penicillium - storage - high temperature drying - air conditioning - ventilation - agricultural research
    Bij 'Tete-a-Tete' zijn er grote partijverschillen in aantasting door Penicillium. PPO-onderzoek geeft aan dat uitval door Penicillium sterk te beperken is door de bollen zo snel mogelijk na rooien bij hoge temperaturen te drogen. De methode van warm drogen heeft zeker toekomst. Wel is van groot belang voor voldoende ventilatie en circulatie te zorgen om vocht tijdig af te voeren
    Goede bolontsmetting hoeft niet duur en milieubelastend te zijn : onderzoek lelie
    Kok, B.J. ; Aanholt, J.T.M. van - \ 2003
    BloembollenVisie 2003 (2003)12. - ISSN 1571-5558 - p. 18 - 19.
    bloembollen - lilium - plantenziekteverwekkende schimmels - penicillium - desinfecteren - milieubescherming - bedrijfsvergelijking in de landbouw - kosten - ornamental bulbs - lilium - plant pathogenic fungi - penicillium - disinfestation - environmental protection - farm comparisons - costs
    De effectiviteit van zeven bolontsmettingsrecepten van zeven preparatiebedrijven tegen Penicillium in lelie worden vergeleken
    Germination inhibitors of fungal spores: identification and mode of action
    Chitarra, G.S. - \ 2003
    Wageningen University. Promotor(en): Frans Rombouts, co-promotor(en): Tjakko Abee; J. Dijksterhuis. - [S.l.] : S.n. - ISBN 9789058089144 - 111
    schimmelsporen - kiemremmers - fusarium culmorum - penicillium - fluorescentiemicroscopie - antimycotica - fungal spores - germination inhibitors - fusarium culmorum - penicillium - fluorescence microscopy - antifungal agents
    Fungi can be found in a wide variety of environments, such as seeds, plants, soil, water, insects, and food products. Fungi and their toxic metabolites cause losses of food products, and diseases in plants and animals, and may have adverse effects on human health. A crucial step in fungalcolonisationand infection is the germination process, subsequently resulting inmycelialgrowth. The aim of this thesis was to study fungal germination and the effects of antifungal compounds on this process usingPenicilliumpaneum conidia andFusariumculmorummacroconidiaas model organisms.

    An antifungal compound produced by Bacillussubtilis YM 10-20 was identified and characterized as aniturin-like compound belonging to theiturinfamily. This compound inhibited fungal germination and growth ofPenicilliumpaneum conidia. Fluorescence probes in combination with flowcytometryand scanning electron microscopy were applied to assess the action of the antifungal compound against spores. Destruction and morphological changes of conidia in the presence of the inhibitor were observed. Theiturin-like compoundpermeabilisedfungal spore membranes and blocked germination. Another compound was identified and characterized fromdense conidia suspensions ofPenicilliumpaneum.A volatile compound(s) were produced under high-density conditions by the conidia themselves. It inhibitedmycelialgrowth of different species of fungi belonging to a variety of genera, suggesting a broad action range.1-Octen-3-ol was identified as a volatile self-inhibitor of spore germination.This compoundinfluenced different developmental processes during the P.paneum life cycle namelymicrocycleconidiationand spore germination.The compound henceforth acts as a fungal hormone in the life cycle of the fungus. The mode of action of 1-octen-3-ol was investigated. It targets the membrane affecting intracellular pH, respiration, and protein synthesis.

    The sequence of events in the germination process ofmulticompartmentFusariumculmorummacroconidiaand the effects of antifungal compounds on this process was investigated by measuring the intracellular pH (pH in) employing fluorescence ratio imaging microscopy (FRIM). During germ tube formation, clear differentiation between the compartments of themacroconidiumwas observed. Germ tubes develop preferably from apical cells and with low frequency from middle compartments. ThepH in varied among different compartments and during different stages of germination.Addition ofantifungals,nystatinandnonanoicacid during germination, affectspH inof the conidia and their differentiation.

    The work described in this thesis highlights the sequence of events in the germination process of fungal spores, notably conidia from Penicilliumpaneum andmulticompartmentmacroconidia fromFusariumculmorum and the effect of antifungal compounds. Detailed insight in the fungal germination process and action of antifungal compounds may contribute to a more efficient control of fungal infection of plants and animals, food spoilage and toxin production.

    Beheersing en bestrijding van Botrytis cinerea en van Penicillium in Euphorbia fulgens
    Wubben, J.P. ; Hazendonk, A. ; Bosker, I. ; Slootweg, C. ; Hoope, M. ten - \ 2002
    Aalsmeer : Praktijkonderzoek Plant & Omgeving, Sector Glastuinbouw (PPO 555) - 29
    euphorbia fulgens - houtachtige planten als sierplanten - plantenziekten - schimmelziekten - botrytis cinerea - penicillium - nederland - euphorbia fulgens - ornamental woody plants - plant diseases - fungal diseases - botrytis cinerea - penicillium - netherlands
    De bloeiwijze van Euphorbia fulgens kent twee belangrijke schimmelbelagers, die problemen in de teelt veroorzaken: Botrytis cinerea en Penicillium. B. cinerea geeft schade in de vorm van smet of pokken, die op de bloemblaadjes verschijnen. Dit zijn kleine donkerbruine/zwarte plekjes van ongeveer 1 mm doorsnede. Deze schimmel geeft met name problemen in de teelt wanneer de luchtvochtigheid hoog is, omdat de schimmel vocht nodig heeft om aantasting te geven. Penicillium komt met name voor in de bloemhartjes van E. fulgens en vormt daar een dicht grijsgroen schimmelpluis. In de bloemhartjes bevindt zich nectar en dit is een goede voedingsbodem voor Penicillium. In de praktijk worden verschillende teeltmaatregelen genomen om aantasting door B. cinerea en Penicillium te beperken. Een aantal bedrijven kiezen ervoor om zo droog mogelijk te telen waardoor B. cinerea minder kans krijgt. Een andere groep probeert juist door het afsproeien van de bloemen de nectar te verwijderen waarbij Penicillium minder kans heeft. Het moge duidelijk zijn dat deze laatste maatregel B. cinerea juist meer kans geeft omdat de luchtvochtigheid in het gewas zal toenemen. In het onderzoek is gekeken naar de beste teeltmaatregel tegen B. cinerea en Penicillium. Daarnaast is onderzocht wat de huidige mogelijkheden voor biologische bestrijding voor B. cinerea en Penicillium zijn
    Extracellular polysaccharides as target compounds for the immunological detection of Aspergillus and Penicillium in food
    Kamphuis, H.J. - \ 1992
    Agricultural University. Promotor(en): F.M. Rombouts; S.H.W. Notermans. - S.l. : Kamphuis - ISBN 9789054850199 - 157
    voedselbesmetting - voedselmicrobiologie - aspergillus - penicillium - polysacchariden - food contamination - food microbiology - aspergillus - penicillium - polysaccharides

    This thesis is devoted to the immunological detection of Aspergillus and Penicillium in food products. More specifically, the immunogenicity, antigenicity, production and structure of the water-soluble extracellular polysaccharides (EPS) of these moulds have been studied, and a latex-agglutination assay, based on the detection of EPS has been developed.

    For the detection of moulds many methods are available, each of them with specific advantages and disadvantages, mostly related to reliability and applicability ( Chapter 2 ).

    An overview of the immunogenicity and antigenicity of EPS produced by moulds is presented in Chapter 3 . The role of β(1,5)-galactofuranoside sequences as epitopes of galactomannans from Aspergillus and Penicillium is documented. Antigenically specific polyclonal antibodies raised against P.digitatum EPS are directed towards β(1,5)-linked galactofuranosyl residues. These antibodies react specifically with EPS from Aspergillus and Penicillium.

    Synthetic tetramers and heptamers of β(1,5)-linked galactofuranosides are conjugated to tetanus toxoid and polyclonal antibodies are raised in rabbits against these synthetic immunogens ( Chapter 4 ). Antibodies obtained after immunisation with the heptamer conjugate possess the same genus specific antigenicity as the antibodies raised against P.digitatum EPS. No reactions are observed with the Penicillium subgenus biverticillium species and species belonging to genera other than Aspergillus and Penicillium . In contrast, antibodies raised against the tetramer conjugate reacted only with six out of 24 tested Aspergillus and Penicillium strains.

    From Glucanex, a Trichoderma harzianum enzyme preparation, an exo-β-D- galactofuranosidase is purified. This enzyme is used to hydrolyse specifically the immunodominant β(1,5)-linked galactofuranosyl residues from Aspergillus and Penicillium EPS. This enzyme alleviates the antigenicity of the EPS completely ( Chapter 5 ).

    Additionally, the reductive cleavage method for determination of the glycosidic bonds revealed that the β(1,5)-linked galactofuranosyl side chains in P.digitatum EPS carry side-chains of β(1,6)-1inked galactofuranosyl residues. These results allowed to propose a new structural model for the antigenically active galactofuranoside side chains of Penicillium galactomannans.

    In Chapter 6 , the production of antigenic EPS by P. aurantiogriseum and P. digitatum has been described under various growth conditions. Antigenic EPS was produced under almost all conditions investigated. However, both P.aurantiogriseum and P.digitatum do not produce antigenic EPS on lactate as the carbon source. Also, P.camemberti isolated from a mould fermented cheese (Camembert) does not produce antigenic EPS on lactate, althought, P.camemberti produces antigenic EPS on other substrates. The monosaccharide composition of the EPS produced by P.aurantiogriseum and P.digitatum under various conditions varies considerably.

    Immunopotent acid-labile β(1,5)-linked galactofuranosyl residues of Aspergillus fumigatus, Aspergillus niger and Penicillium digitatum EPS were acid-hydrolysed ( Chapter 7 ). Antibodies are raised against these acid-treated extracellular polysaccharides. It was supposed that these acid-treated EPS preparations would elicit antibodies with a broader specificity, making them useful in the detection of nearly all or all moulds occurring in food products. However, the antibodies obtained are more species specific and are generally directed to glucosyl and/or mannosyl residues of the EPS.

    Antibodies raised against P.digitatum EPS are used for the development of a rapid and reliable latex-agglutination assay for the detection of Aspergillus and Penicillium in food and feed ( Chapter 8 ). The reliability of the assay is enhanced by using a synthetic epitope, a tetramer of β(1,5)-Iinked galactofuranosides. With this tetramer false-positive results can easily be recognised.

    Finally, in Chapter 9 the applicability of the developed latex-agglutination assay is tested in both comparative and collaborative studies. The significance of extracellular polysaccharides produced by moulds for the detection of moulds in food and feed is discussed.

    Mechanism of resistance in Penicillium italicum to fungicides which inhibit sterol 14alpha-demethylation
    Guan, J. - \ 1992
    Agricultural University. Promotor(en): J. Dekker; M.A. de Waard. - S.l. : Guan - 133
    penicillium - gewasbescherming - fungiciden - resistentie tegen pesticiden - penicillium - plant protection - fungicides - pesticide resistance

    The fungitoxic action of azole fungicides is based on inhibition of cytochrome P450-dependent sterol 14α-demethylase (cytochrome P450 14DM ) activity. This thesis presents results of studies on various potential mechanisms of resistance to these sterol 14α-demethylation inhibitors (DMIs) in DMI-resistant isolates of the fungal plant pathogen Penicillium italicum. In chapters 2, 4, 6, 7 and 8 results of studies on four potential mechanisms of resistance are described. In chapters 3 and 5 two important developments in experimental procedures which were essential to test one of the potential mechanisms of resistance, decreased affinity of the target enzyme, are presented.

    In chapter 2, sterol composition of wild-type isolate W 5 and isolates E 300-3 (low resistance), H 17 (medium resistance), I 33 and J 4 (high resistance) is described. Results showed that in all isolates ergosterol was the major sterol. Inhibition of ergosterol biosynthesis in W 5 was observed at a low concentration of imazalil (0.01 μg m1 -1), while in the resistant isolates much higher concentrations were necessary to achieve the inhibitory effect. Inhibition of ergosterol biosynthesis was accompanied by accumulation of 24-methylenedihydrolanosterol. These results proved that the same target site for imazalil is present in all isolates and that the mechanism of resistance in this fungus is not related to absence of the target enzyme or the presence of a nonfunctional one.

    Chapter 3 describes an important achievement in studying in vitro enzymes involved in sterol biosynthesis in filamentous plant pathogenic fungi. A novel method to obtain a cell-free extract of P. italicum which was active in synthesizing ergosterol from [ 14C]mevalonate is presented and may serve as a model to study similar processes in other filamentous plant pathogens. The in vitro synthesis of ergosterol in cell-free extracts of P. italicum accounted for about 26% of total non- saponifiable lipids synthesized. The inhibitory effect of various test compounds (DMI fungicides and less-toxic imazalil analogues) on in vitro ergosterol biosynthesis was investigated. IC 50 values (concentrations which inhibit incorporation of radioactivity into ergosterol by 50%) of the highly toxic DMI fungicides imazalil, itraconazole, ketoconazole, penconazole and propiconazole ranged from 6.5 ± 0.5 x 10 -9to 1.7 + 0.7 x 10 -11M. This indicates that DMI fungicides are very potent inhibitors of sterol 14α- demethylase activity in cell-free extracts of the fungus. Less-toxic imazalil analogues had much higher IC 50 values, suggesting that these compounds have a significantly lower potency to inhibit sterol 14α-demethylase activity. The test method used to study ergosterol biosynthesis in the wild-type isolate W 5 could also be applied to DMI-resistant isolates E 300-3 , HP and I 33 , It was noted that the quality of cell-free extracts of wild-type and resistant isolates differed slightly. Nevertheless, IC 50 values of imazalil for inhibition of in vitro ergosterol biosynthesis were not significantly different in wild-type and all DMI-resistant isolates (Chapter 4). This indicates that sterol 14α-demethylase of sensitive and resistant isolates has a similar sensitivity to imazalil. Therefore, reduced affinity of the target enzyme to DMIs in resistant isolates will not play a major role as a mechanism of resistance.

    In Chapter 5, a method to isolate cytochrome P450 isozymes from wildtype isolate W 5 is described. Interaction between the heterocyclic nitrogen atom of the test compounds (DMI-fungicides and less-toxic imazalil analogues) and the oxidized heme iron atom of the isozymes could be demonstrated by difference spectrophotometry (type II spectra). However, the magnitude of the type II spectra did not correlate with toxicity of the test compounds. A difference in the ability of carbon monoxide (CO) to displace the test compound from reduced cytochrome P450 isozymes was observed. It appeared that the less-toxic imazalil analogues were immediately displaced while the displacement of various DMI fungicides gradually occurred in time. This suggests that the binding affinity of the test compounds to cytochrome P450 isozymes did correlate to some degree with fungitoxicity of the test compounds. However, inconsistent results were observed for DMI fungicides with a large N 1-substituent, like itraconazole and ketoconazole.

    Cytochrome P450 isozymes were also isolated from DMI-resistant isolates E 300-3 , H 17 , I 33 and J 4 (Chapter 6). However, the procedure for isolate H 17 , I 33 and J 4 had to be slightly modified in order to isolate proper P450 isozymes. The necessary modification of the isolation procedure suggests that the resistant isolates H 17 , I 33 and J 4 differ in some way, possibly in cell wall composition, from that of the wild-type isolate. Maximum and minimum absorbance in type II spectra of P450 isozymes of isolates H 17 , I 33 and J 4 shifted to higher wavelengths as compared with those of the wild-type isolate W 5 and low-resistant isolate E 300-3 , Maximum absorbance in CO spectra of P450 isozymes of isolates H 17 , I 33 and J 4 also shifted to higher wavelengths. Imazalil, itraconazole and ketoconazole were more readily displaced by CO from reduced P450 isozymes of isolates H 17 , I 33 and J 4 than from those of wild-type isolate W 5 and isolate E300-3 (Chapter 6). These results suggest that P450 isozymes of isolates H 17 , I 33 and J 4 had a relatively lower affinity to DMI fungicides. However, due to various factors which hamper a proper interpretation of the results, conclusions derived from spectrophotometric assays may not be reliable enough to compare affinity of cytochrome P450 14DM to DMIs in different isolates. Hence, these results do not provide sound evidence to reject the conclusion that sterol 14α-demethylase of wild-type and DMI-resistant isolates have a similar sensitivity to DMIs (Chapter 4).

    In Chapter 7, results of experiments on metabolism of imazalil in wildtype and DMI-resistant isolates are presented. It appeared that all isolates metabolized imazalil to its putative 2,3-dihydroxypropyloxy analogue (R42243), which was much less fungitoxic. DMI-resistant isolates showed cross resistance to miconazole which has a similar structure as imazalil except for the propenyloxy side chain. Therefore, it was concluded that metabolism of imazalil in P. italicum is not involved as a mechanism of resistance.

    Fungicide accumulation studies indicated that the low-resistant isolate E300-3 accumulated a significantly lower level of imazalil and fenarimol than the wild-type isolate W 5 (Chapter 8). The low level of accumulation of the fungicides may be responsible for the relatively low degree of resistance to these fungicides in this isolate. Reduced accumulation of fenarimol may be caused by an increased energy-dependent efflux of the fungicide. However, this mechanism is less obvious for imazalil. Accumulation of both fungicides is probably not mediated by the plasma membrane potential. Medium- and high-resistant isolates H 17 , I 33 and J 4 accumulated similar levels of imazalil and fenarimol as the low-resistant isolate E 300-3 . This suggests that the mechanism of resistance in medium- and high-resistant isolates is not caused by differential accumulation of the DMI fungicides as compared to the lowresistant isolate E 300-3 . However, it might be that differential accumulation of the DMIs between low- and high-resistant isolates is present, but masked by a relatively high background adsorption of the fungicides to mycelium and therefore, not detected with the test method used.

    From the results of the studies summarized above, it is concluded that the mechanism of resistance in P. italicum to DMIs relates to factors which prevent the fungicides from reaching the target site. Further studies should elucidate which mechanism is relevant in this respect.

    Effects of imazalil on sterol composition of sensitive and DMI-resistant isolates of Penicillium italicum.
    Guan, J. ; Kerkenaar, A. ; Waard, M.A. de - \ 1989
    Netherlands Journal of Plant Pathology 95 (1989)Suppl. 1. - ISSN 0028-2944 - p. 73 - 86.
    fungiciden - penicillium - pesticidenwerking - ongediertedodende eigenschappen - resistentie tegen pesticiden - pesticiden - gewasbescherming - fungicides - penicillium - pesticidal action - pesticidal properties - pesticide resistance - pesticides - plant protection
    Resistance to benomyl and some chemically related compounds in strains of Penicillium species
    Bollen, G.J. - \ 1971
    Wageningen : [s.n.] (Mededeling / Laboratorium voor phytopathologie no. 275) - 7
    fungiciden - resistentie tegen pesticiden - plantenziekteverwekkende schimmels - gewasbescherming - penicillium - fungicides - pesticide resistance - plant pathogenic fungi - plant protection
    Recherches sur la maladie, due al Metarrhizium anisopliae chez le criquet pelerin
    Veen, K.H. - \ 1968
    Wageningen University. Promotor(en): J. de Wilde; A.J.P. Oort. - Wageningen : Veenman - 77
    acrididae - sprinkhanen - insecten - plantenplagen - biologische bestrijding - schimmels - organismen ingezet bij biologische bestrijding - penicillium - dierziekten - dierpathologie - acrididae - locusts - insects - plant pests - biological control - fungi - biological control agents - penicillium - animal diseases - animal pathology
    Introduction
    The data from the literature concerning insect killing Deuteromycetes are discussed. Stress is laid on many fundamental problems and on gaps in the scientific observations.

    Nomenclature
    It appears from a bibliographic survey that Metarrhizium brunneum PETCH, Metarrhizium album PETCH and Metarrhizium anisopliae (METSCHNIKOFF) SOROKIN most probably represent a single species. The conidial lengths divide the fourteen isolated strains into two groups; these conform to the 'forma major' and the 'forma minor' of JOHNSTON (1915). Because of the supposed absence of original type material of M. anisopliae the author fixed a neotype and deposited it in the Centraal Bureau voor Schimmelcultures at Baarn (The Netherlands).

    Isolation, development and growth, storing
    Conidiospores of M. anisopliae, isolated from 14 species of insects, were cultivated on cherry-agar, on the medium according to MARTIN (1950) or on a special isolation medium (VEEN & FERRON, 1966). In the last medium, actidione (cycloheximide) and chloramphenicol is added in order to prevent infections by bacteria and fungi. The fungus sporulates excellently in surface cultures when the Chapoteaut peptone composition is used as nutrient medium. The mass production of conidiospores has been carried out on boiled sterile rice. The formation of blastospores has been tested in shake cultures according to the Adamek (1967) method; this has been done with five strains of forma major and five of forma minor. Generally a concentration of 10 8-10 9blastopores per ml. is obtained after 96 hours; one strain, however, sporulated only poorly.

    The strains could be successfully stored at + 4°C for at least 20 months after culturing on egg-yolk coagulated at 80°C. Lyophilization of conidial suspensions in skimmed milk did not affect the germinating capacity of the spores for six months.

    List of host insects
    Scrutiny of the literature revealed that at least 204 species of insects are susceptible to M.anisopliae under natural conditions. These are mostly Coleoptera of which the terricolous species predominate.

    Germination and development of germtubes
    Conidiospores of M.anisopliae germinate only in contact with water. Intersegmental folds of the host Schistocerca gregaria FORSK. prevented direct observations, but germination of the conidiospores on exposed parts of the cuticle apparently are not affected by the transpiration of the insect. Epicuticular lipids of fifth stage larvae evoke conidial germination, in contrast to beeswax. The composition of the nutrient medium used for sporulation, may likewise exercise great influence on germination of spores when transferred to an extraction of epicuticular lipids of locusts; the various strains of M.anisopliae however behave differently. Both in vivo and in vitro the germinated fungus forms structures analogous to 'appressoria'. But in vitro only rarely penetration tubes originate beneath the 'appressoria'. Also the latter structures are not always essential to the development of penetration tubes.

    Penetration and infection
    Histological sections show that the infection of second instar locust larvae by conidia of a race of M. anisopliae isolated from S. gregaria takes place after about 90 hours. The site of penetration of the hyphae is the mouth cavity, mainly the proximal part of the maxillary palps. The ambient relative humidity has no influence on this process.

    Penetration of exposed integument, however, occurs only if a water film is present.

    Experiments on third instar larvae have shown that the fungus has no preference for specific areas of the integument (as for instance the inter-segmental membranes) for penetration. Any area of the integument can be penetrated. It has been observed that both chemical and mechanical activities play a part in the penetration process.

    Phagocytosis of the fungus by haemocytes was never observed.

    Infection also takes place when conidiospores are injected in the rectum, but obviously this is of no importance under normal conditions when infection through the mouth prevails.

    Probably, blastospores are formed in the places where the penetrating mycelium. comes into contact with circulating haemolymph.

    The last stage of the disease
    When the insect dies, the amount of fungus material in the body is often relatively insignificant. Shortly before death the insects often behave abnormally. These observations suggest an action of toxins. Therefore, the toxicity of culture filtrates of ten strains of M. anisopliae was assayed on caterpillars of Galleria mellonella L. and larvae of S.gregaria. Considerable differences in activity between the strains were found. However, we were unable to establish a correlation between the production of toxins of the different strains and the degree of virulence for S. gregaria.

    Sporulation
    When locust larvae are kept in very high relative humidity, bacterial infections with a variable mortality are not uncommon. These unfavorable conditions obviously change the physiological condition of the insects. The early stages of an infection by M. anisopliae are also characterized by a reduced vitality of the host. In many cases blastospores as well as bacteria are found side by side in the haemolymph, even before death. A competition between these microorganisms then occurs after death.

    Under these conditions the sporulation of the fungus is inhibited, though to a different degree in different strains. This is also confirmed by the observation that bacterial colonies mainly (Aeromonas sp.) may locally suppress the growth of the fungus on a Sabouraud culture medium in Petri dishes. When few bacteria are present, sporulation on insect bodies only takes place in a high relative humidity. A relative humidity of 93 % is already below the limit necessary for sporulation.

    Concentration effects
    Experiments with spores in quantities sufficient to kill second instar hoppers have shown that considerable differences in activity exist within the twelve strains studied. The LD 50 of a strain isolated from Polyphylla olivieri CAST. has been determined. The inclination of the regression line representing the relation between the logarithm of the dose and the mortality in probits is fairly large and resembles the values obtained from the insect killing bacterium Bacillus thuringiensis BERL. In similar experiments the food of the hoppers was sprayed with a suspension of spores. These experiments have been performed on first, second and third instar larvae. It appears that the susceptibility of the hoppers to the fungus decreases only slightly during aging, but the incubation period becomes longer in older larvae. Dosage experiments may also be performed per os with blastospores from shake cultures.

    CONCLUSIONS

    In the past the use of insect killing materials, containing M. anisopliae gave only rather unsatisfactory results. However, from the present investigation it appears that the fungus possesses some features which may render it useful to control the desert locust especially larvae of the first and second instar.

    As it has been established that infection in this species may occur via the mouth, it is possible to induce a fungal infection even in air of a very low humidity, as is often found in the habitat of this insect species. The chance of inducing an epizootic, however, seems to be limited because sporulation of the fungus, essential for a spreading of the disease, requests a very high relative humidity.

    To determine the possibilities of oral infection in other insect species, it is necessary to study the conditions under which M. anisopliae becomes infectious. Also the doses of spores which are needed to cause high mortality within a short time should be determined. Only after this has been done it will be possible to derive the conditions for practical application of this biological control agent.

    Of course technical factors and production costs will be decisive for its eventual use.

    Fundamental research is needed in order to select highly virulent strains and to maintain their virulence or to increase it by physical or chemical methods. It is also essential to investigate the specificity of the various strains to harmful and useful insects, as well as their possible effects on vertebrates, though the latter seems unlikely in view of the existing literature.

    Technical possibilities and the economics of mass-production will finally be decisive for the use.

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