Botrytis cinera kan in vrijwel alle gewassen vóórkomen, zowel buiten als in bedekte teelten. Deze 'grauwe schimmel', kan alle delen van een plant aantasten: stengel, bladeren, vruchten en bloemen. Het is een typsiche zwakteparasiet, omdat hij vooral voorkomt op wonden en verouderde plantendelen.

Elke winter is het weer raak met uitval door de Botrytis cinerea in de broeierij van tulp. De schimmel slaat vooral toe in de potgrondteelt en de pottenbroei. Nu het broeierijseizoen weer voor de deur staat, is het goed om de symptomen en de maatregelen op een rij te zetten.

Onderzoek bij Wageningen UR geeft inzicht hoe en waarom de schimmel een plant aantast. Deze kennis kan worden gebruikt bij het ontwikkelen van resistente gewassen, bemestingsadviezen en biologische bestrijding. Voorlopig moet de praktijk Botrytis voorkomen door een goede bedrijfshygiëne en de juiste kasklimaatregeling

Botrytis cinerea is a fungal pathogen of more than 200 hosts including a wide variety of economically important crops. Although many ecological and physiological studies on this destructive pathogen have been reported, not much is known about the molecular basis of the interaction of this pathogen with its various host plants. This thesis describes the use of molecular techniques to study the genetic variation and pathogenicity of B. cinerea.Genetic variation among ten strains of B. cinerea was studied by RAPID analysis. Strains appeared to be highly similar and could not be grouped according to their host, suggesting that host specialization does not occur in B. cinerea . Segregation analysis of RAPID markers in generative progeny collected from ordered ascospore octads, predominantly revealed segregation ratios of 1:1. Occasionally, a 1:3 segregation ratio, or the appearance and disapperance of RAPID markers were observed. These unexpected observations were explained by the heterokaryotic association of presumably polyploid nuclei.An important role for cutinase was suggested in penetration of undamaged host tissue. To investigate the relevance of cutinase in more detail, cloning of the cutinase gene was an essential step. Molecular strategies such as heterologous screening and immunoscreening of libraries and a PCR based cloning strategy were employed without success. Purification of the enzyme, partial determination of its amino acid sequence and the design of cutinase specific primers finally led to the cloning and identification of the corresponding gene ( cutA ). A second putative esterase was co-purified and the encoding gene (ekdA) was isolated as well.Using a cutA promoter - GUS reporter gene fusion, expression of the cutinase gene was studied in planta . Conidia of transformants harbouring the reporter construct and germinating on gerbera flowers and tomato fruits, contained GUS activity, indicating that the cutinase gene is expressed during penetration of host tissue by B. cinerea . However, disruption of the single copy cutA gene in a haploid strain of B. cinerea , showed that this cutinase is not required for successful infection of gerbera flowers and tomatoes. The ability of cutinase A-deficient mutants to infect and to develop disease was unaltered compared to the wild type strain. These results suggest that B. cinerea employs other strategies to penetrate undamaged host tissue. Production of other cutinases, generation of oxygen radicals by glucose oxidase or mechanical penetration by formation of appressoria might be involved.

C ell w all d egrading e nzyme s (CWDEs) secreted by microbial plant pathogens have been suggested to function as virulence factors. Evidence that particular bacterial CWDEs contribute to virulence has emerged in the last two decades. Targeted gene replacement of different genes encoding CWDEs resulted in mutants with reduced virulence on a number of host plants. Similar molecular genetic approaches in plant pathogenic fungi have, until recently, been unsuccessful in elucidating a role for fungal CWDEs in pathogenesis. This thesis describes molecular genetic analyses of CWDEs secreted by the necrotrophic plant pathogenic fungus Botrytis cinerea , the causal agent of gray mould.From literature it was known that B. cinerea secretes many CWDEs when grown in liquid culture. The number of CWDE encoding genes present in the B. cinerea genome was unknown and detailed expression studies were lacking. In order to fill this knowledge gap we used the following strategy:Cloning of genes encoding CWDEsStudy of the expression of CWDE genes both in liquid cultures and in plantaTargeted deletion of CWDE genes that have expression patterns that indicate a function in the infection processChapter 1 introduces the research area and gives an outline of the thesis. It describes a model of the chemical and structural composition of the plant cell wall and reviews various classes of microbial CWDEs. It summarises previously published data on the role of bacterial and fungal CWDEs in pathogenesis in general and on the CWDEs secreted by B. cinerea in particular. B. cinerea has a wide host range but prefers hosts that contain high amounts of pectin. Therefore the focus was on endo p oly g alacturonases (endoPGs), enzymes that cleave homogalacturonan, a major constituent of pectin.In order to study gene expression of B. cinereain planta , it was essential to develop a standardised inoculation procedure that enables reproducible infections both in time and space. The development of this inoculation procedure for tomato leaves is described in Chapter 2. The expression of two fungal genes and a number of plant PR-protein genes was investigated in time course experiments performed at two different incubation temperatures.Subsequently, we set out to clone the genes of interest, analysed their expression and studied the effect in pathogenesis by targeted gene replacement. The genes were isolated by hybridisation with heterologous probes. The first gene that was cloned and characterised, Bcpg 1, is constitutively expressed. Targeted replacement of this gene resulted in a mutant with reduced virulence on apple fruits and tomato (Chapter 3). Subsequently, five additional endoPG genes were isolated (Chapter 4). The gene products were compared with other fungal endoPGs and it was shown that the members of the B. cinereaBcpg gene family fall into at least three distinct monophyletic groups (Chapter 4).The members of the endoPG gene family, denoted as Bcpg 1-6, are differentially expressed in liquid cultures that differed in carbon source or pH (Chapters 4). The constitutive expression pattern of Bcpg 1, as found in Chapter 3, was further confirmed. Bcpg 2 is expressed under all circumstances tested except when B. cinerea is grown in glucose-containing medium at low pH. Bcpg 3 is expressed at low ambient pH. Bcpg 4 is induced by the pectin breakdown end-product galacturonic acid, and is repressed by glucose. Bcpg 5 expression can be induced by a yet unknown factor present in apple pectin. Bcpg 6 is, like Bcpg 4, induced by galacturonic acid but is, unlike Bcpg 4, not repressed by glucose. The expression of the endoPG gene family enables the fungus to degrade pectate in a flexible manner. It enables the fungus to respond to environmental signals like nutrient availability and pH.The expression of the endoPG gene family during infection of tomato leaf, broad bean leaf, apple fruit and courgette fruit was studied (Chapter 5). Expression of the genes in planta is differential and most expression patterns can be explained by the results of expression studies in liquid cultures. Bcpg 1 is expressed in all host tissues tested, whereas expression of Bcpg 2 is evident in tomato, broad bean and courgette. Bcpg 3 and Bcpg 5 are expressed in apple fruit. Bcpg 4 and Bcpg 6 are expressed in all host tissues tested.Chapter 6 discusses the results in a broader context. It is hypothesised that, besides Bcpg 1, additional members of the Bcpg gene family contribute to virulence, albeit likely under specific circumstances. It is suggested that fungal CWDEs can play a role in plant pathogenesis but that this role also strongly depends on the lifestyle of the fungus. It is postulated that B. cinerea depends strongly on endoPGs for successful infection. The research described in this thesis may lead to novel disease control strategies that rely on P oly G alacturonase I nhibiting P rotein (PGIP) expression in transgenic host plants.

After some introductory words on flax, different forms of the parasite Botrytis cinerea Pers. ex Fr. were compared. Use of differences in production of organic acids as done by van Beyma Thoe Kingma were not a satisfactory distinction between formae lini of Botrytis. The M, Sc and Sp growth forms, few isolated interforms, B. bifurcata Miller and B. allii Munn. all produced significant amounts of citric acid; the amount depended on isolation, temperature, nutrient medium and amount of CaC0 3 in the medium. Although the species was polyphagous, where it grew as a parasite was related to its growth form.Stages discussed of the disease in flax were the juvenile phase (caused by infected seed, spread through the soil to other plants), an intermediate phase (during accelerated growth of the flax, the stem base was attacked by spores of the juvenile phase), and the flowering phase (spores from elsewhere or from the interphase).Other topics were: environmental influences encouraging grey mould during the phases; ways of treating flax seed, research on seed infection (comparison of the Ulster and filter-paper method); differences in infectivity with environment; and finally chemical disinfection of seed and its toxicity to the plant.

Botrytis cinerea Pers. ex Fr., the causal agent of grey mould, is one of the most ubiquitous plant pathogens. The fungus is of high economic importance in various major crops and during transport and storage of agricultural products. Protectant fungicides such as chlorothalonil, dichlofluanid, folpet or thiram are widely used for disease control. Since their introduction in the 1960S/1970s, systemic fungicides such as the benzimidazoles or dicarboximides have been used extensively. However, their effectivity is severely hampered by a rapid development of resistance to these fungicides.Antifungal activity of N1-substituted azoles was discovered in the late 1960s. Since then, a large number of azole derivatives have been developed as agricultural fungicides and antimycotics. The mode of action of these azoles is based on inhibition of the cytochrome P450-dependent sterol 14α-demethylase (P450 14DM ), an enzyme of the sterol pathway. By now, sterol demethylation inhibitors (DMIs) comprise about 35 commercial products and represent the most important group of systemic fungicides. DMI fungicides are commonly applied in control of rusts, powdery mildews and scabs. Only few of them are registered for control of B. cinerea . This is ascribed to a limited field performance for which the reasons are not evident. A replacement of dicarboximides or benzimidazoles by DMI fungicides would be attractive, since DMIs have a number of advantages over other fungicides including a relatively low resistance risk.The aim of the study described in this thesis is to identify factors involved in the limited field performance of DMI fungicides towards B. cinerea . The study is restricted to the largest group of DMIs, the triazoles. Before presenting results obtained in this study a literature review on the biology and control of B. cinerea , the mode of action and mechanisms involved in selective fungitoxicity of DMI fungicides, and factors responsible for discrepancies in laboratory and field pesticide performance is given ( chapter 1 ). Biological activity of triazoles towards B. cinerea was investigated in vitro with cell-free assays ( chapters 3 - 4 ) and toxicity assays ( chapters 3 - 7) and in vivo on different hosts ( chapter 5 ).The first step in the research presented in this thesis was the development of a cell-free assay for sterol synthesis from the model fungus Penicillium italicum ( Moniliaceae ) according to a method described for Aspergillus fumigatus ( chapter 2 ). Subsequently, the method developed was adopted for Botrytis cinerea ( chapter 3). This assay was used to study the relationship between chemical structure and biological activity of commercial and experimental triazoles and stereoisomers of cyproconazole,SSF-109 and tebuconazole towards B. cinerea ( chapter 4 ). On basis of these experiments intrinsic inhibitory activity of triazoles towards P450 14DM of the target pathogen was determined. in following experiments, factors which influence In vivo activity or field performance were Investigated. In vivo activity of triazole fungicides towards B. cinerea was tested on foliar-sprayed tomato plants and diptreated grape berries, and compared with that of selected benzimidazoles and dicarboximides ( chapter 5 ). in this context was also studied whether biological compounds could specifically antagonize activity of triazoles ( chapter 5 ). Variation in triazole sensitivity of the pathogen population was studied for field isolates (121) of B. cinerea collected during 1970 - 1992 in Europe and israel ( chapter 6 ). in this survey less sensitive populations were detected. A putative mechanism of resistance to DMI fungicides in field isolates with a relatively low sensitivity to DMIs was studied and compared with that operating in laboratory-generated DMI-resistant mutants ( chapter 7 ). Effects of inhibitors of mitochondrial respiration and multisite-inhibiting fungicides on accumulation of tebuconazole were tested to evaluate their potency as candidate compounds in synergistic mixtures with DMIs ( chapter 7 ). The development of synergistic mixtures may improve biological activity of DMI fungicides in control of B. cinerea.

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.

Op basis van het literatuuronderzoek, de gegevens van de plantmonitoren en de gegevens van de praktijkproeven zijn we meer te weten gekomen over een aantal factoren die de nectarvorming beïnvloeden. Helaas leren dezelfde gegevens ons ook dat het voorkomen of afremmen van nectarvorming niet mogelijk is. Zelfs bij de meest optimale groeiomstandigheden vormt de plant nectar in de bloem. Wel kunnen we door zo gelijkmatig mogelijke groeiomstandigheden te realiseren de vorming wel iets reguleren, maar niet meer dan dat

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 dr.ir. M.A. de Waard, leerstoelgroep Fytopathologie, Wageningen Universiteit

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 dr.ir. M.A. de Waard, leerstoelgroep Fytopathologie, Wageningen Universiteit

De schimmel Ulocladium atrum is geselecteerd als een antagonist van Botrytis cinerea. De ecologische eigenschappen van deze antagonist en de toepassingen op bovengrondse plantendelen worden in dit artikel beschreven. Gegevens bij de bijgaande figuren: 1) Effect van Ucladium atrum op de grauwe schimmel in druif; 2) Schade aan Botrytis cinerea in tomaat en de bestrijding hiervan door U. atrum; 3) Schade van Botrytis cinerea in cyclaam en de bestrijding hiervan door U. atrum

De schimmel Ulocladium atrum is geselecteerd als een antagonist van Botrytis cinerea. De ecologische eigenschappen van deze antagonist en de toepassingen op bovengrondse plantendelen worden in dit artikel beschreven. Gegevens bij de bijgaande figuren: 1) Effect van Ucladium atrum op de grauwe schimmel in druif; 2) Schade aan Botrytis cinerea in tomaat en de bestrijding hiervan door U. atrum; 3) Schade van Botrytis cinerea in cyclaam en de bestrijding hiervan door U. atrum

Naar aanleiding van een vraag uit de praktijk is nagegaan in welke mate Botrytis een probleem is in aardappelen en wat er aan gedaan kan worden. Geconcludeerd mag worden dat de ziekte zich vooral aan het einde van het seizoen laat zien en heeft daardoor weinig invloed op de opbrengst. Extra gevoelige rassen zijn niet vastgesteld. Er zijn in Nederland geen middelen toegelaten ter bestrijding van deze schimmelziekte in aardappelen.

Op het eerste gezicht lijken Botrytis, bijen en hommels niets met elkaar te maken te hebben, maar toch is er een verband. Honingbijen en hommels kunnen namelijk botrytisinfecties helpen bestrijden of voorkomen. Veel plantpathogene schimmels, waaronder botrytis, infecteren de planten via de bloemen. Als er een andere, voor bloemen ongevaarlijke schimmel, bacterie of gist in de bloem groeit, is daarmee Botrytis te remmen of te voorkomen. Een reportage over deze schimmel en de verspreiding van BCA's door bijen en hommels

ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporters are two major classes of proteins involved in drug resistance. ABC transporter proteins are primary transporters that use the energy generated by ATP hydrolysis to transport drugs over membranes, while MFS transport proteins are secondary transporters that use the proton motive force as an energy source. The substrate range of both transporters is very broad and may include ions, amino acids, peptides, sugars, secondary metabolites, and drugs. The goal of this thesis was to identify ABC and MFS transporter genes from Botrytis cinerea, which are involved in transport of sterol demethylation inhibitor (DMI) fungicides, and to discover compounds, which can modulate the activity of such transporters. Such modulators may be useful in practice to counteract resistance development to DMIs.An overview of characteristics of B. cinerea , commercial fungicides used in control of the pathogen, modes of action of botryticides, and resistance development to fungicides that inhibit sterol biosynthesis is described in Chapter 1. This chapter also describes the relevance of ABC and MFS transporters in fungicide resistance and the importance of fungicide mixtures to delay resistance development.The DMI fungicide oxpoconazole, developed by Ube industries, Ltd., is introduced in Chapter 2. This fungicide is effective against diseases that are commonly controlled by DMIs, such as rusts and scabs. In addition, it is effective against grey mould caused by B. cinerea under field condition. B. cinerea is known for its ability to acquire resistance to fungicides easily. ABC and MFS transporters may play a role in such a resistance development. The physiological functions of these transporters may be to cope with natural plant toxins since the pathogen has an extraordinary wide host range.Chapter 3 describes fourteen ABC and three MFS transporter genes from B. cinerea . Two of the ABC transporters were described previously and the others were identified in an EST library of the fungus. Their role in DMI resistance was investigated by studying basal and induced expression in wild-type and DMI-resistant strains of B. cinerea. From this screen, it appeared that BcatrD was the most probable ABC transporter gene encoding a putative DMI transporter since basal expression in three isolates correlated with sensitivity to DMIs. Induced expression after treatment with DMIs also correlated with sensitivityto DMIs. The MFS transporter gene Bcmfs1 might encode another DMI transporter since its basal expression in DMI resistant strains was also higher than that in the wild-type strain.The functional analysis of BcatrD and Bcmfs1 is described in Chapters 4 and 5, respectively. This was achieved by phenotyping of gene replacement and overexpression mutants generated from the haploid wild-type strain B05.10. Replacement and overexpression mutants of BcatrD displayed an increased and decreased sensitivity to DMIs, respectively. Overexpression mutants of Bcmfs1 also exhibited a decreased sensitivity to these fungicides while Bcmfs1 replacement mutants showed similar sensitivity to DMIs as compared to the wild-type. To clarify the role of Bcmfs1 in DMI-sensitivity in more detail, we constructed a double replacement mutant of BcatrD and Bcmfs1 . The double replacement mutant was more sensitive to DMIs than the single replacement mutant of BcatrD. These results suggest that BcatrD functions as a major DMI transporter and that Bcmfs1 is of minor importance. Accumulation of oxpoconazole by germlings of the mutants is also described in Chapter 4 and 5. Accumulation of oxpoconazole was transient in time as observed before for other DMIs and other filamentous fungi. The transient accumulation pattern is the result of passive influx and inducible active efflux of fungicides by transporters. The initial accumulation (20 min after the addition of oxpoconazole) level of oxpoconazole in the strains tested correlated with sensitivity to oxpoconazole. The steady state levels (more than 60 min after the addition of oxpoconazole) of accumulation by BcatrD and Bcmfs1 replacement mutants were higher than that by the wild-type isolate. These results indicate that BcatrD and Bcmfs1 mediate the sensitivity of B. cinerea to oxpoconazole by reducing the accumulation of the fungicide in mycelial cells.

De grauwe schimmel Botrytis cinerea is een parasiet van een groot aantal plantensoorten en kan zowel kiemplanten als alle bovengrondse delen van groeiende planten aantasten. Vaak heet de aantasting 'grauwe schimmel', maar de aantasting kan ook andere namen hebben. Botrytis is in bijna alle grote teelten een ziekte waarop alle technieken losgelaten worden. De problemen van Botrytis in de Gerbera-teelt worden nader uitgelegd. DLV Plant heeft recentelijk onderzoek uitgevoerd naar Botrytis in de aardbeienteelt en de bewaarfase van hortensia.