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Op 8 januari 2007 promoveerde Jerome Custers aan Wageningen Universiteit op het proefschrift getiteld “Engineering disease resistance in plants”. Promotor was Prof. Dr.Ir.P.J.G.M. de Wit, verbonden aan de leerstoelgroep Fytopathologie, Wageningen Universiteit. Co-promotor was Dr. M.H. Stuiver, Head of Genomics, BASF Plant Science GmbH, Limburgerhof, Duitsland. Het werk beschreven in dit proefschrift werd uitgevoerd bij Syngenta Mogen B.V. (voorheen MOGEN International NV) in Leiden. Een reportage over gen-om-gen resistentie genereren, plantenpathogenen, afweerreacties tegen infecties en interacties tussen de gastheer en het plantenpathogeen

Op 27 september 2004 promoveerde Marco Kruijt aan Wageningen Universiteit op het proefschrift getiteld 'Molecular evolution of Cladosporium fulvum disease resistance genes in wild tomato'. De evolutie van het Cf-9 gen in de wilde tomatensoort L.pimpinellifolium is in detail bestudeerd. Naast het Cf-9 gen blijkt nog een resistentiegen, het 9DCgen, aanwezig. Het Cf-9 gen is ouder dan het 9DC gen en komt in verschillende wilde tomatensoorten voor. Hieruit blijkt ondermeer dat C. fulvum waarschijnlijk al een pathogeen van de 'oertomaat' was

Op 20 juni 2011 promoveerde Hendrik Rietman aan Wageningen University (WUR) op zijn proefschrift getiteld: “Putting the Phytophthora infestans genome sequence at work; multiple novel avirulence and potato resistance gene candidates revealed”. Zijn promotor, Prof. Dr. Richard G. F. Visser, en co-promotor, Dr. Ir. Vivianne G. A. A. Vleeshouwers, zijn beiden verbonden aan de leerstoelgroep Plantenveredeling van de WUR, waar ook het onderzoek werd uitgevoerd. Tevens is er samengewerkt met de leerstoelgroep Fytopathologie (WUR), het Sainsbury Lab en het James Hutton Instituut (beiden gevestigd in het Verenigd Koninkrijk). Financiering was afkomstig van het ‘Parapluplan Phytophthora’ en WUR Plantenveredeling.

Op 4 oktober 2004 promoveerde Maarten de Kock aan de Wageningen Universiteit op het proefschrift getiteld 'Recognition of the Cladosporium fulvum Ecp2 elicitor in tomato and non-host plants'. De resultaten van dit promotieonderzoek vormen een wetenschappelijke basis voor verdere bestudering van de co-evolutie tussen planten en pathogenen en maken een voorzichtige voorspelling van de duurzaamheid van resistentiegenen mogelijk

Op 20 juni 2011 promoveerde Hendrik Rietman aan Wageningen University (WUR) op zijn proefschrift getiteld: “Putting the Phytophthora infestans genome sequence at work; multiple novel avirulence and potato resistance gene candidates revealed”. Zijn promotor, Prof. Dr. Richard G. F. Visser, en co-promotor, Dr. Ir. Vivianne G. A. A. Vleeshouwers, zijn beiden verbonden aan de leerstoelgroep Plantenveredeling van de WUR, waar ook het onderzoek werd uitgevoerd. Tevens is er samengewerkt met de leerstoelgroep Fytopathologie (WUR), het Sainsbury Lab en het James Hutton Instituut (beiden gevestigd in het Verenigd Koninkrijk). Financiering was afkomstig van het ‘Parapluplan Phytophthora’ en WUR Plantenveredeling.

Op 20 juni 2011 promoveerde Hendrik Rietman aan Wageningen University (WUR) op zijn proefschrift getiteld: “Putting the Phytophthora infestans genome sequence at work; multiple novel avirulence and potato resistance gene candidates revealed”. Zijn promotor, Prof. Dr. Richard G. F. Visser, en co-promotor, Dr. Ir. Vivianne G. A. A. Vleeshouwers, zijn beiden verbonden aan de leerstoelgroep Plantenveredeling van de WUR, waar ook het onderzoek werd uitgevoerd. Tevens is er samengewerkt met de leerstoelgroep Fytopathologie (WUR), het Sainsbury Lab en het James Hutton Instituut (beiden gevestigd in het Verenigd Koninkrijk). Financiering was afkomstig van het ‘Parapluplan Phytophthora’ en WUR Plantenveredeling.

An attempt to balance selection intensity and conservation of genetic diversity was made in the national program for breeding for scrapie resistance in the Netherlands. The ARR allele is associated with resistance to scrapie. In the Netherlands a breeding program was started on a voluntary basis in 2001 and starting 2004 the use of ARR/ARR rams was obligatory. In some rare breeds there were so few ARR/ARR rams that the risk of high inbreeding and associated loss of genetic diversity would be unacceptably high. Based on computer simulations predicting the inbreeding rates for several breeds exemptions on the obligatory breeding program were allowed. In this paper the ARR frequencies and associated inbreeding rates are analyzed as an indication of loss in genetic diversity during the years of scrapie selection in two breeds: the “Zwartbles schaap” (Black-blazed sheep) and the “Melkschaap” (Friesian-Zealand Milksheep).

An attempt to balance selection intensity and conservation of genetic diversity was made in the national program for breeding for scrapie resistance in the Netherlands. The ARR allele is associated with resistance to scrapie. In the Netherlands a breeding program was started on a voluntary basis in 2001 and starting 2004 the use of ARR/ARR rams was obligatory. In some rare breeds there were so few ARR/ARR rams that the risk of high inbreeding and associated loss of genetic diversity would be unacceptably high. Based on computer simulations predicting the inbreeding rates for several breeds exemptions on the obligatory breeding program were allowed. In this paper the ARR frequencies and associated inbreeding rates are analyzed as an indication of loss in genetic diversity during the years of scrapie selection in two breeds: the “Zwartbles schaap” (Black-blazed sheep) and the “Melkschaap” (Friesian-Zealand Milksheep).

Iron is fundamental to the biology of eukaryotic cells since it plays a key role in many metabolic functions. Iron concentrations are tightly regulated, for example by ferritin, because excessive iron leads to tissue damage. Iron cannot cross cellular membranes directly and most cells acquire iron from the iron transporting protein transferrin (Tf), via transferrin receptors. During nutritional immunity the body reacts with a metabolic adjustment in order to render important nutrients unavailable to invading microorganisms. However, pathogens also have evolved a range of mechanisms to acquire iron from the host (chapter 1). In the study described in this thesis, we used a natural host-parasite model of common carp (Cyprinus carpio L.) infected with Trypanoplasma borreli, a protozoan kinetoplastid, extracellular blood parasite of carp to get more insight in the competition for iron between host and parasite. Transferrin of common carp is highly polymorphic with several alleles identified according to differences in electrophoretic mobility. We studied the implications of Tf polymorphisms for iron binding and modulation of immune function. We performed a series of challenge experiments infecting five genetically different, commercially exploited carp lines with T. borreli. Our results indicated that Tf genotype may influence the susceptibility to pathogens. We observed a significant association of the DD genotype of Tf with low parasitaemia in two resistant carp lines (Polish ‘R2’ and ‘K’), but a reverse association in the most susceptible carp line ‘D’ (chapter 2). We also showed that variation in resistance to T. borreli could be controlled by sex-related genetic factors. Examination of parasite growth in vitro, in culture media supplemented with 3% serum taken from fish with different Tf genotypes, showed a faster decrease in number of parasites in media supplemented with serum from DD-typed animals (chapter 2). In general, pathogens also have mechanisms to acquire iron from the host. We developed a method for Tf depletion of carp serum using specific antibodies to carp Tf, and compared T. borreli multiplication and survival in the presence or absence of Tf in vitro. Parasites were dying in medium containing Tf-depleted serum, which clearly showed that Tf is essential for parasite growth and multiplication (chapter 3). We isolated two allelic forms of carp Tf (alleles D and G) to purity using rivanol precipitation and ion-exchange chromatography (chapter 5). We showed that parasite growth in vitro could be reconstituted by the addition of purified Tf to Tf-depleted serum (chapter 3). We observed differences in T. borreli multiplication and survival in culture media containing different sera typed differently for Tf genotypes (chapter 3). We identified four complete coding sequences for common carp Tf alleles C, D, F and G, and confirmed the overall similarity of the carp Tf three-dimensional structure to Tfs of other species. We could show that carp Tf differs significantly in critical iron-binding sites in the N-lobe of the molecule, as compared to other non-cyprinid fish species (chapter 4). The substitution of a majority of the iron-coordinating residues in the Nlobe indeed seems to affect the ability to bind iron, which may be compensated for by higher serum concentrations of Tf (chapter 7). Comparison of constitutive gene expression of two Tf alleles D and G showed a comparably high gene expression level in liver and small but consistent differences in gene expression for allele D over allele G in other immunologically important organs (chapter 4). Our data suggest that the allelic polymorphism is not related to differences in iron binding and/or binding to the host Tf receptor but could be linked with other factors, such as competition for iron with pathogens (chapter 4). Transferrin itself may also exert effects that are not directly linked with maintaining iron levels and Tf cleavage products have been shown to stimulate macrophages to produce large amounts of nitric oxide (NO). To study the induction of NO in carp head kidney-derived macrophages, we isolated two allelic forms of carp Tf (alleles D and G) to purity and showed that the level of activation of macrophages by Tf was different for the D and G allele (chapter 5). Differences in NO levels induced could be related to different cleavage forms of the two alleles D and G, as shown by Western blot, confirming that full-length Tf cannot induce NO. The D-type Tf cleavage products induced significantly higher nitric oxide (NO) production than cleavage products of Gtype Tf. (chapter 5). Transferrin uptake by trypanosome parasites involves Tf binding to a receptor. The TfR-Tf complex then is internalised and transported to lysosomes, where Tf is proteolytically degraded. We described the cloning and sequencing of a cathepsin L-like cysteine proteinase from T. borreli and production of a recombinant and biologically active enzyme (chapter 6). We demonstrated that the T. borreli cysteine proteinase is able to digest host transferrin. Likely, Tf cleavage fragments are released from the trypanosomes while iron would remain parasite-associated, possibly contributing to thepathogenicity of the parasite by inducing high amounts of NO in carp macrophages (chapter 7). Our study dealt with different aspects of Tf polymorphism, discussing the role of Tf in immunity of common carp and the influence of allelic polymorphism on competition for iron between host and pathogen (chapter 7). Further investigations should shed more light on the selective advantage of particular alleles to provide a basis for incorporating Tf as a genetic marker in marker-assisted selection programmes for increased resistance to diseases. This could contribute to improved survival of carp kept under semiintensive farming systems in ponds.

Iron is fundamental to the biology of eukaryotic cells since it plays a key role in many metabolic functions. Iron concentrations are tightly regulated, for example by ferritin, because excessive iron leads to tissue damage. Iron cannot cross cellular membranes directly and most cells acquire iron from the iron transporting protein transferrin (Tf), via transferrin receptors. During nutritional immunity the body reacts with a metabolic adjustment in order to render important nutrients unavailable to invading microorganisms. However, pathogens also have evolved a range of mechanisms to acquire iron from the host (chapter 1). In the study described in this thesis, we used a natural host-parasite model of common carp (Cyprinus carpio L.) infected with Trypanoplasma borreli, a protozoan kinetoplastid, extracellular blood parasite of carp to get more insight in the competition for iron between host and parasite. Transferrin of common carp is highly polymorphic with several alleles identified according to differences in electrophoretic mobility. We studied the implications of Tf polymorphisms for iron binding and modulation of immune function. We performed a series of challenge experiments infecting five genetically different, commercially exploited carp lines with T. borreli. Our results indicated that Tf genotype may influence the susceptibility to pathogens. We observed a significant association of the DD genotype of Tf with low parasitaemia in two resistant carp lines (Polish ‘R2’ and ‘K’), but a reverse association in the most susceptible carp line ‘D’ (chapter 2). We also showed that variation in resistance to T. borreli could be controlled by sex-related genetic factors. Examination of parasite growth in vitro, in culture media supplemented with 3% serum taken from fish with different Tf genotypes, showed a faster decrease in number of parasites in media supplemented with serum from DD-typed animals (chapter 2). In general, pathogens also have mechanisms to acquire iron from the host. We developed a method for Tf depletion of carp serum using specific antibodies to carp Tf, and compared T. borreli multiplication and survival in the presence or absence of Tf in vitro. Parasites were dying in medium containing Tf-depleted serum, which clearly showed that Tf is essential for parasite growth and multiplication (chapter 3). We isolated two allelic forms of carp Tf (alleles D and G) to purity using rivanol precipitation and ion-exchange chromatography (chapter 5). We showed that parasite growth in vitro could be reconstituted by the addition of purified Tf to Tf-depleted serum (chapter 3). We observed differences in T. borreli multiplication and survival in culture media containing different sera typed differently for Tf genotypes (chapter 3). We identified four complete coding sequences for common carp Tf alleles C, D, F and G, and confirmed the overall similarity of the carp Tf three-dimensional structure to Tfs of other species. We could show that carp Tf differs significantly in critical iron-binding sites in the N-lobe of the molecule, as compared to other non-cyprinid fish species (chapter 4). The substitution of a majority of the iron-coordinating residues in the Nlobe indeed seems to affect the ability to bind iron, which may be compensated for by higher serum concentrations of Tf (chapter 7). Comparison of constitutive gene expression of two Tf alleles D and G showed a comparably high gene expression level in liver and small but consistent differences in gene expression for allele D over allele G in other immunologically important organs (chapter 4). Our data suggest that the allelic polymorphism is not related to differences in iron binding and/or binding to the host Tf receptor but could be linked with other factors, such as competition for iron with pathogens (chapter 4). Transferrin itself may also exert effects that are not directly linked with maintaining iron levels and Tf cleavage products have been shown to stimulate macrophages to produce large amounts of nitric oxide (NO). To study the induction of NO in carp head kidney-derived macrophages, we isolated two allelic forms of carp Tf (alleles D and G) to purity and showed that the level of activation of macrophages by Tf was different for the D and G allele (chapter 5). Differences in NO levels induced could be related to different cleavage forms of the two alleles D and G, as shown by Western blot, confirming that full-length Tf cannot induce NO. The D-type Tf cleavage products induced significantly higher nitric oxide (NO) production than cleavage products of Gtype Tf. (chapter 5). Transferrin uptake by trypanosome parasites involves Tf binding to a receptor. The TfR-Tf complex then is internalised and transported to lysosomes, where Tf is proteolytically degraded. We described the cloning and sequencing of a cathepsin L-like cysteine proteinase from T. borreli and production of a recombinant and biologically active enzyme (chapter 6). We demonstrated that the T. borreli cysteine proteinase is able to digest host transferrin. Likely, Tf cleavage fragments are released from the trypanosomes while iron would remain parasite-associated, possibly contributing to thepathogenicity of the parasite by inducing high amounts of NO in carp macrophages (chapter 7). Our study dealt with different aspects of Tf polymorphism, discussing the role of Tf in immunity of common carp and the influence of allelic polymorphism on competition for iron between host and pathogen (chapter 7). Further investigations should shed more light on the selective advantage of particular alleles to provide a basis for incorporating Tf as a genetic marker in marker-assisted selection programmes for increased resistance to diseases. This could contribute to improved survival of carp kept under semiintensive farming systems in ponds.

Op 8 januari 2007 promoveerde Jerome Custers aan Wageningen Universiteit op het proefschrift getiteld “Engineering disease resistance in plants”. Promotor was Prof. Dr.Ir.P.J.G.M. de Wit, verbonden aan de leerstoelgroep Fytopathologie, Wageningen Universiteit. Co-promotor was Dr. M.H. Stuiver, Head of Genomics, BASF Plant Science GmbH, Limburgerhof, Duitsland. Het werk beschreven in dit proefschrift werd uitgevoerd bij Syngenta Mogen B.V. (voorheen MOGEN International NV) in Leiden. Een reportage over gen-om-gen resistentie genereren, plantenpathogenen, afweerreacties tegen infecties en interacties tussen de gastheer en het plantenpathogeen

Op 8 januari 2007 promoveerde Jerome Custers aan Wageningen Universiteit op het proefschrift getiteld “Engineering disease resistance in plants”. Promotor was Prof. Dr.Ir.P.J.G.M. de Wit, verbonden aan de leerstoelgroep Fytopathologie, Wageningen Universiteit. Co-promotor was Dr. M.H. Stuiver, Head of Genomics, BASF Plant Science GmbH, Limburgerhof, Duitsland. Het werk beschreven in dit proefschrift werd uitgevoerd bij Syngenta Mogen B.V. (voorheen MOGEN International NV) in Leiden. Een reportage over gen-om-gen resistentie genereren, plantenpathogenen, afweerreacties tegen infecties en interacties tussen de gastheer en het plantenpathogeen

Tomato early blight (EB) caused by the fungus Alternaria solani is a field disease with a worldwide distribution, including Indonesia. The disease is currently controlled using frequent applications of fungicides. The use of resistant cultivar would be an attractive way to reduce fungicide application. The aims of the research are to support breeding of EB resistance cultivars for the Indonesian market, by identifying EB resistance genes effective in Indonesia and developing markers for MAS.In Chapter 1 the literature on A. solani, EB and resistance is reviewed. On the fungal side pathogenicity and genetic variation have been widely investigated. However, the existence of different physiological races has not been convincingly demonstrated or disproved. Reliable methods of screening are available for use in resistance breeding. Intensive screening of tomato accessions worldwide has shown that strong resistance is not available in the cultivated tomato but only in wild species. Resistance to A. solani is expressed quantitatively and is polygenically inherited. For that reason classical breeding has not been able to achieve high levels of resistance, and undesirable traits from the donors have been introgressed as well. Recently QTLs have been mapped in a few resistance sources, which could facilitate transferring such quantitative resistance genes and circumvent the problem of unfavourable linkage drag once markers tightly linked to the QTLs have been obtained.In Chapter 2 a resistance test method involving droplet inoculation is described, which is an adaptation of an existing but hardly-used technique. This method has advantages in comparison with the more widely used spray inoculation method, including a clear distinction between lesions caused by A. solani and necroses of 1eaf loss due to other causes, and an objective measurement of damage in contrast to subjective scoring. In the droplet inoculation method leaflets of intact plants are inoculated with droplets of an A. solani conidial suspension in water or an agar solution. Lesions are assessed quantitatively by measuring the length and the perpendicular width. In this way objective and accurate assessments, which are prerequisites for QTL analysis, can be achieved. This inoculation method was used to screen a collection of 54 tomato accessions for resistance to an Indonesian A. solani isolate,S. arcanum LA2157 is highly resistant to A. solani in the glasshouse screening tests. The genetics of the resistance in this wild relative of tomato was further studied using a QTL mapping approach (Chapter 3). The mapping population consisted of 176 F2 plants. A linkage map consisting of 12 linkage groups covering 1179 cM was based on 379 markers (33 SSR, 21 SNP and 316 AFLP markers). All linkage groups could be assigned to the 12 tomato chromosomes. About half of the markers showed deviation from the expected 1:2:1 segregation ratio. The F2 population was phenotyped in a glasshouse in Netherlands with an Indonesian isolate of A. solani. EB resistance was evaluated with respect to lesion size (LS) and related parameters (relative area under the lesion expansion curve [RAULEC] and percentage of small lesions [PSmL]). The derived F3 lines (156) were tested in a field in Indonesia. Percentage of EB index (PEBI) was assessed at six times and relative area under the disease progress curve (RAUDPC) was calculated. A total of six QTLs with a range of LOD scores 3.6 to 16.4 were mapped on chromosomes 1, 2, 5, 6, 7 and 9. Three of the QTLs showed effects in both tests despite differences in experimental method and observed traits. For the first time, three QTLs for resistance affecting the development of stem lesions, another disease symptom caused by the fungus on the main and secondary stems of the plant, were identified, which completely overlapped with QTL regions for early blight resistance. Two QTLs on chromosomes 2 and 9, which explained 7 to 16% of the phenotypic variation for EB resistance and 31% for stem lesion resistance, are recommended to be used in tomato breeding programs for resistance to A. solani.

Tomato early blight (EB) caused by the fungus Alternaria solani is a field disease with a worldwide distribution, including Indonesia. The disease is currently controlled using frequent applications of fungicides. The use of resistant cultivar would be an attractive way to reduce fungicide application. The aims of the research are to support breeding of EB resistance cultivars for the Indonesian market, by identifying EB resistance genes effective in Indonesia and developing markers for MAS.In Chapter 1 the literature on A. solani, EB and resistance is reviewed. On the fungal side pathogenicity and genetic variation have been widely investigated. However, the existence of different physiological races has not been convincingly demonstrated or disproved. Reliable methods of screening are available for use in resistance breeding. Intensive screening of tomato accessions worldwide has shown that strong resistance is not available in the cultivated tomato but only in wild species. Resistance to A. solani is expressed quantitatively and is polygenically inherited. For that reason classical breeding has not been able to achieve high levels of resistance, and undesirable traits from the donors have been introgressed as well. Recently QTLs have been mapped in a few resistance sources, which could facilitate transferring such quantitative resistance genes and circumvent the problem of unfavourable linkage drag once markers tightly linked to the QTLs have been obtained.In Chapter 2 a resistance test method involving droplet inoculation is described, which is an adaptation of an existing but hardly-used technique. This method has advantages in comparison with the more widely used spray inoculation method, including a clear distinction between lesions caused by A. solani and necroses of 1eaf loss due to other causes, and an objective measurement of damage in contrast to subjective scoring. In the droplet inoculation method leaflets of intact plants are inoculated with droplets of an A. solani conidial suspension in water or an agar solution. Lesions are assessed quantitatively by measuring the length and the perpendicular width. In this way objective and accurate assessments, which are prerequisites for QTL analysis, can be achieved. This inoculation method was used to screen a collection of 54 tomato accessions for resistance to an Indonesian A. solani isolate,S. arcanum LA2157 is highly resistant to A. solani in the glasshouse screening tests. The genetics of the resistance in this wild relative of tomato was further studied using a QTL mapping approach (Chapter 3). The mapping population consisted of 176 F2 plants. A linkage map consisting of 12 linkage groups covering 1179 cM was based on 379 markers (33 SSR, 21 SNP and 316 AFLP markers). All linkage groups could be assigned to the 12 tomato chromosomes. About half of the markers showed deviation from the expected 1:2:1 segregation ratio. The F2 population was phenotyped in a glasshouse in Netherlands with an Indonesian isolate of A. solani. EB resistance was evaluated with respect to lesion size (LS) and related parameters (relative area under the lesion expansion curve [RAULEC] and percentage of small lesions [PSmL]). The derived F3 lines (156) were tested in a field in Indonesia. Percentage of EB index (PEBI) was assessed at six times and relative area under the disease progress curve (RAUDPC) was calculated. A total of six QTLs with a range of LOD scores 3.6 to 16.4 were mapped on chromosomes 1, 2, 5, 6, 7 and 9. Three of the QTLs showed effects in both tests despite differences in experimental method and observed traits. For the first time, three QTLs for resistance affecting the development of stem lesions, another disease symptom caused by the fungus on the main and secondary stems of the plant, were identified, which completely overlapped with QTL regions for early blight resistance. Two QTLs on chromosomes 2 and 9, which explained 7 to 16% of the phenotypic variation for EB resistance and 31% for stem lesion resistance, are recommended to be used in tomato breeding programs for resistance to A. solani.

Op 27 september 2004 promoveerde Marco Kruijt aan Wageningen Universiteit op het proefschrift getiteld 'Molecular evolution of Cladosporium fulvum disease resistance genes in wild tomato'. De evolutie van het Cf-9 gen in de wilde tomatensoort L.pimpinellifolium is in detail bestudeerd. Naast het Cf-9 gen blijkt nog een resistentiegen, het 9DCgen, aanwezig. Het Cf-9 gen is ouder dan het 9DC gen en komt in verschillende wilde tomatensoorten voor. Hieruit blijkt ondermeer dat C. fulvum waarschijnlijk al een pathogeen van de 'oertomaat' was

Jaarlijks valt 10% van de seringenstruiken uit als gevolg van aantasting door Verticillium dahliae. Dit rapport beschrijft allereerst het onderzoek waarin een besmettingsmethode voor jonge seringenstruikjes is ontwikkeld. Daarna is in de periode 2003-2005 begonnen met de selectie van weinig voor Verticillium vatbare onderstammen om deze in combinatie met een cultivar in de praktijk te gebruiken voor de teelt van trekseringen

Potato is the most important non-cereal crop in the world. Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating disease of potato. In the mid-191h century, P. infestans attacked the European potato fields and this resulted in a widespread famine in Ireland. Late blight remains the No.l constraint to potato production and causes a yearly multi-billion US$ loss globally. In Europe and North America, late blight con trol heavily relies on the use of chemicais, which is hardly affordable to farmers in developing countries and also raises considerabie environmental concerns in the developed countries.Use of host resistance is ecologically the most sustainable way of disease management. Disease resistance (R) genes exist in a wide range of wild species of the genera Solanum. One wild species, Solanum demissum, became the donor of most characterized R genes due to its crossability with the cultivated species S. tuberosum. However, these race-specific genes (Rl-Rll) didn't enable satisfactory protection under the current deployment scheme. Breeders either turned to other wild species or adopted a so-called 'R-gene-free' approach to explore quantitative resistance. Unfortunately, neither alternative offered a solution to late blight control and did not result in commercial release of cultivars with durable resistance.At the end of the 20th century, biological research entered the genomics era, landmarked by the Human Genome Project and by the Arabidopsis and rice genome sequencing initiatives. Genomics also became the new frontier of research in potato and P. infestans. This scientific development is deepening and broadening our understanding of the biology of the host and the pathogen and is facilitating isolation of key genes involved in the interaction. The genetically modified organism (GMO) strategy allows a much more efficient application of these genes than time-consuming conventional breeding. This thesis deals with the isolation, characterization, and deployment of host R genes with the expectation to achieve an ecologically and economically sound control of late blight.The potato-P.infestans interaction follows the gene-for-gene model, that is, resistance only occurs when a host R gene and its corresponding avirulence (Avr) gene in the pathogen are both present. Several disease testing methods have been developed for determination of the gene-for-gene interaction between potato andP. infestans. In vitro inoculation was developed as a quick, space-effective, and accurate assay (Chapter 2). The method exploits the amenability of potato for tissue culture and the suitability of the in vitro environment for late blight disease development.ltsspecificity and reliability was confirmed by comparison with the well-established detached-Ieaf assay. Currently, in vitro inoculation is routinely used in phenotyping of segregating populations, resistance testing of transformants for functional complementation, and screening of new R genes in a wide range of wild germplasms.The investigation of host resistance was focused on the R3 complex locus on the distal part of chromosome I\. The R3 complex locus segregated in a potato mapping population, which was used to construct the potato ultra-high dense (UHD) map saturated with over 10,000 amplified fragment length polymorphism (AFLP) markers. Using a population of 1748 plants, we constructed a high-resolution genetic map at the R3 complex locus. The combination of fine-mapping and accurate disease testing with specific P. infestans isolates resulted in the unexpected discovery that the R3 complex locus is composed of two functionally distinct genes, R3a and R3b, which are 0.4 cM apart and have both been introgressed from S. demissum (Chapter 3). Each gene was localized into a genetic interval of 0.25 cM, providing the starting point for map-based cloning.Plant R gene families undergo fast evolution, resulting in considerable intraand inter-specific variation. Plant disease resistance (R) loci frequently lack synteny between related species of cereals and crucifers but appear to be positionally well conserved in the Solanaceae. Comparative genomics provides a tooi to utilize the exponentially increasing sequence information from model plants to clone agronomically important genes from less studied crop species. We were keen to investigate whether this tooi can enable new R gene cloning by a case study. The comparative study revealed that the potato late blight R3 locus and the tomato Fusarium wilt 12 locus were derived from an ancestrallocus involved in plant innate immunity. We adopted alocal RGA approach using DNA sequences of the 12 gene to isolate the R3a gene (Chapter 4).l2 and R3a share 88% and 83% identities at the DNA and protein level, respectively. R3a is a member of the R3 complex locus. Comparative physical mapping disclosed that the potato R3 complex locus underwent a significant expansion after divergence from tomato without disruption of the flanking colinearity. This expansion resulted in an increase in the number of R genes and functional diversification.lnterestingly, the differential evolution of the ancient R locus in the two closely related species is well correlated with the contrasting evolutionary potentials of the pathogens with which 12 and R3 interact.Fusarium oxysporum is a soilborne fungus with low genotype diversity, whereas the late blight pathogen P. infestans is notorious in its ability to move and mutate. An intriguing question is whyS. demissum.aspecies that appears to contain only easily-broken R genes, can display durable resistance at the population level in its natural environment. The polymorphism of parasite recognition capacity in a host population will restrict most isolates of the parasite to grow on most hosts. Allelism is an efficient way of creating recognition polymorphism. We made another unexpected discovery that the R3 complex locus has very high allelic diversity and that the R5 -R 11 resistant specificities all contain a distinct allelic version (Chapter 5). Sequence exchange between alleles and diversifying selection are the major driving forces of this allelic recogntion polymorphism to P. infestans. Remarkably, the genomic structure of the R3 complex locus favors the creation of new resistance specificity by reshuffling of elements from the two clusters (R3a and R3b). The multiple allelism of the R3 complex locus may be a natural mechanism of S. demissum to suppress late blight epidemics and should be mimicked in resistance breeding. We suggest that R-gene polyculture via the GMO approach should be the future paradigm of R gene deployment in late blight controI.Potato breeding for late blight resistance was one of the earliest mankind practices in combating plant pathogens by means of genetic improvement but the disease has not been controlled by resistance breeding so faro The non-durable nature of S. demissum R genes apparently disappointed most breeders and the blame of easily-broken R genes even led to the unsuccessful R-gene-free approach. However, from an evolutionary and ecological point of view, single R genes can never defeat pathogens such as P. infestans with extremely high evolutionary risk. We propose that the community rethinks its strategy of R gene deployment in late blight disease management. R gene monoculture is obviously not recommended. R gene pyramiding is currently practiced by breeders but this strategy is basically the same as R gene monoculture since it creates uniformity in host resistance specificity in the field, which will be eventually broken by the fast-evolving pathogen. R gene polyculture should be the strategy of the future. However, we should soberly realize that host resistance might be high enough for survival of the plant population but it alone might never offer a protection that meets the economical threshold. Integrated pest management should include R gene polyculture as the central element, cultivation measures, and limited chemical applications To provide this central element, we need to clone more R genes. Our discovery of the multiple allelism of the R3 potato late blight resistance complex and molecular characterization of one of its allelic versions will offer a possibility to a dozen R genes in the near future and then to deploy them in a potato field using marker-free GMO techniques.

Op 4 oktober 2004 promoveerde Maarten de Kock aan de Wageningen Universiteit op het proefschrift getiteld 'Recognition of the Cladosporium fulvum Ecp2 elicitor in tomato and non-host plants'. De resultaten van dit promotieonderzoek vormen een wetenschappelijke basis voor verdere bestudering van de co-evolutie tussen planten en pathogenen en maken een voorzichtige voorspelling van de duurzaamheid van resistentiegenen mogelijk

Op 27 september 2004 promoveerde Marco Kruijt aan Wageningen Universiteit op het proefschrift getiteld 'Molecular evolution of Cladosporium fulvum disease resistance genes in wild tomato'. De evolutie van het Cf-9 gen in de wilde tomatensoort L.pimpinellifolium is in detail bestudeerd. Naast het Cf-9 gen blijkt nog een resistentiegen, het 9DCgen, aanwezig. Het Cf-9 gen is ouder dan het 9DC gen en komt in verschillende wilde tomatensoorten voor. Hieruit blijkt ondermeer dat C. fulvum waarschijnlijk al een pathogeen van de 'oertomaat' was

Potato is the most important non-cereal crop in the world. Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating disease of potato. In the mid-191h century, P. infestans attacked the European potato fields and this resulted in a widespread famine in Ireland. Late blight remains the No.l constraint to potato production and causes a yearly multi-billion US$ loss globally. In Europe and North America, late blight con trol heavily relies on the use of chemicais, which is hardly affordable to farmers in developing countries and also raises considerabie environmental concerns in the developed countries.Use of host resistance is ecologically the most sustainable way of disease management. Disease resistance (R) genes exist in a wide range of wild species of the genera Solanum. One wild species, Solanum demissum, became the donor of most characterized R genes due to its crossability with the cultivated species S. tuberosum. However, these race-specific genes (Rl-Rll) didn't enable satisfactory protection under the current deployment scheme. Breeders either turned to other wild species or adopted a so-called 'R-gene-free' approach to explore quantitative resistance. Unfortunately, neither alternative offered a solution to late blight control and did not result in commercial release of cultivars with durable resistance.At the end of the 20th century, biological research entered the genomics era, landmarked by the Human Genome Project and by the Arabidopsis and rice genome sequencing initiatives. Genomics also became the new frontier of research in potato and P. infestans. This scientific development is deepening and broadening our understanding of the biology of the host and the pathogen and is facilitating isolation of key genes involved in the interaction. The genetically modified organism (GMO) strategy allows a much more efficient application of these genes than time-consuming conventional breeding. This thesis deals with the isolation, characterization, and deployment of host R genes with the expectation to achieve an ecologically and economically sound control of late blight.The potato-P.infestans interaction follows the gene-for-gene model, that is, resistance only occurs when a host R gene and its corresponding avirulence (Avr) gene in the pathogen are both present. Several disease testing methods have been developed for determination of the gene-for-gene interaction between potato andP. infestans. In vitro inoculation was developed as a quick, space-effective, and accurate assay (Chapter 2). The method exploits the amenability of potato for tissue culture and the suitability of the in vitro environment for late blight disease development.ltsspecificity and reliability was confirmed by comparison with the well-established detached-Ieaf assay. Currently, in vitro inoculation is routinely used in phenotyping of segregating populations, resistance testing of transformants for functional complementation, and screening of new R genes in a wide range of wild germplasms.The investigation of host resistance was focused on the R3 complex locus on the distal part of chromosome I\. The R3 complex locus segregated in a potato mapping population, which was used to construct the potato ultra-high dense (UHD) map saturated with over 10,000 amplified fragment length polymorphism (AFLP) markers. Using a population of 1748 plants, we constructed a high-resolution genetic map at the R3 complex locus. The combination of fine-mapping and accurate disease testing with specific P. infestans isolates resulted in the unexpected discovery that the R3 complex locus is composed of two functionally distinct genes, R3a and R3b, which are 0.4 cM apart and have both been introgressed from S. demissum (Chapter 3). Each gene was localized into a genetic interval of 0.25 cM, providing the starting point for map-based cloning.Plant R gene families undergo fast evolution, resulting in considerable intraand inter-specific variation. Plant disease resistance (R) loci frequently lack synteny between related species of cereals and crucifers but appear to be positionally well conserved in the Solanaceae. Comparative genomics provides a tooi to utilize the exponentially increasing sequence information from model plants to clone agronomically important genes from less studied crop species. We were keen to investigate whether this tooi can enable new R gene cloning by a case study. The comparative study revealed that the potato late blight R3 locus and the tomato Fusarium wilt 12 locus were derived from an ancestrallocus involved in plant innate immunity. We adopted alocal RGA approach using DNA sequences of the 12 gene to isolate the R3a gene (Chapter 4).l2 and R3a share 88% and 83% identities at the DNA and protein level, respectively. R3a is a member of the R3 complex locus. Comparative physical mapping disclosed that the potato R3 complex locus underwent a significant expansion after divergence from tomato without disruption of the flanking colinearity. This expansion resulted in an increase in the number of R genes and functional diversification.lnterestingly, the differential evolution of the ancient R locus in the two closely related species is well correlated with the contrasting evolutionary potentials of the pathogens with which 12 and R3 interact.Fusarium oxysporum is a soilborne fungus with low genotype diversity, whereas the late blight pathogen P. infestans is notorious in its ability to move and mutate. An intriguing question is whyS. demissum.aspecies that appears to contain only easily-broken R genes, can display durable resistance at the population level in its natural environment. The polymorphism of parasite recognition capacity in a host population will restrict most isolates of the parasite to grow on most hosts. Allelism is an efficient way of creating recognition polymorphism. We made another unexpected discovery that the R3 complex locus has very high allelic diversity and that the R5 -R 11 resistant specificities all contain a distinct allelic version (Chapter 5). Sequence exchange between alleles and diversifying selection are the major driving forces of this allelic recogntion polymorphism to P. infestans. Remarkably, the genomic structure of the R3 complex locus favors the creation of new resistance specificity by reshuffling of elements from the two clusters (R3a and R3b). The multiple allelism of the R3 complex locus may be a natural mechanism of S. demissum to suppress late blight epidemics and should be mimicked in resistance breeding. We suggest that R-gene polyculture via the GMO approach should be the future paradigm of R gene deployment in late blight controI.Potato breeding for late blight resistance was one of the earliest mankind practices in combating plant pathogens by means of genetic improvement but the disease has not been controlled by resistance breeding so faro The non-durable nature of S. demissum R genes apparently disappointed most breeders and the blame of easily-broken R genes even led to the unsuccessful R-gene-free approach. However, from an evolutionary and ecological point of view, single R genes can never defeat pathogens such as P. infestans with extremely high evolutionary risk. We propose that the community rethinks its strategy of R gene deployment in late blight disease management. R gene monoculture is obviously not recommended. R gene pyramiding is currently practiced by breeders but this strategy is basically the same as R gene monoculture since it creates uniformity in host resistance specificity in the field, which will be eventually broken by the fast-evolving pathogen. R gene polyculture should be the strategy of the future. However, we should soberly realize that host resistance might be high enough for survival of the plant population but it alone might never offer a protection that meets the economical threshold. Integrated pest management should include R gene polyculture as the central element, cultivation measures, and limited chemical applications To provide this central element, we need to clone more R genes. Our discovery of the multiple allelism of the R3 potato late blight resistance complex and molecular characterization of one of its allelic versions will offer a possibility to a dozen R genes in the near future and then to deploy them in a potato field using marker-free GMO techniques.

Op 4 oktober 2004 promoveerde Maarten de Kock aan de Wageningen Universiteit op het proefschrift getiteld 'Recognition of the Cladosporium fulvum Ecp2 elicitor in tomato and non-host plants'. De resultaten van dit promotieonderzoek vormen een wetenschappelijke basis voor verdere bestudering van de co-evolutie tussen planten en pathogenen en maken een voorzichtige voorspelling van de duurzaamheid van resistentiegenen mogelijk

In het kader van het onderzoeksprogramma 'Biologische productiesystemen in de akkerbouw en vollegrondsgroenteteelt' is de afgelopen vier jaar onderzoek gedaan naar de mogelijkheden die veredeling kan bieden om het wortelstelsel van uien te verbeteren. Dit lijkt mogelijk door specifieke soortkruisingen met stengelui (Japanse bladui) en Allium roylei, met als bijkomend voordeel dat naast de selectie op een beter type wortelstelsel ook geselecteerd kan worden op resistentie tegen de bladschimmels valse meeldauw en bladvlekkenziekte. Een opvallend onderzoeksresultaat was het effect van arbusculaire mycorrhiza (AM) op het verbeteren van het wortelstelsel en de productiviteit van stengelui, en mogelijk ook ui

In het kader van het onderzoeksprogramma 'Biologische productiesystemen in de akkerbouw en vollegrondsgroenteteelt' is de afgelopen vier jaar onderzoek gedaan naar de mogelijkheden die veredeling kan bieden om het wortelstelsel van uien te verbeteren. Dit lijkt mogelijk door specifieke soortkruisingen met stengelui (Japanse bladui) en Allium roylei, met als bijkomend voordeel dat naast de selectie op een beter type wortelstelsel ook geselecteerd kan worden op resistentie tegen de bladschimmels valse meeldauw en bladvlekkenziekte. Een opvallend onderzoeksresultaat was het effect van arbusculaire mycorrhiza (AM) op het verbeteren van het wortelstelsel en de productiviteit van stengelui, en mogelijk ook ui

Ondernemen met biodiversiteit? Wat kan een agrarisch ondernemer daarmee? In dit werkboek wordt getracht daar antwoord op te geven voor ondernemers in de veehouderij, de akkerbouw, de vollegrondsgroenteteelt en de fruitteelt. Tegelijkertijd worden suggesties gedaan hoe een agrariër met biodiversiteit kan ondernemen en wat dat voor het bedrijf betekent. Dit is een werkboek voor alle soorten en maten van grondgebonden bedrijven. Zowel gangbare, geïntegreerde als biologische bedrijven kunnen het gebruiken

Ondernemen met biodiversiteit? Wat kan een agrarisch ondernemer daarmee? In dit werkboek wordt getracht daar antwoord op te geven voor ondernemers in de veehouderij, de akkerbouw, de vollegrondsgroenteteelt en de fruitteelt. Tegelijkertijd worden suggesties gedaan hoe een agrariër met biodiversiteit kan ondernemen en wat dat voor het bedrijf betekent. Dit is een werkboek voor alle soorten en maten van grondgebonden bedrijven. Zowel gangbare, geïntegreerde als biologische bedrijven kunnen het gebruiken

Ondernemen met biodiversiteit? Wat kan een agrarisch ondernemer daarmee? In dit werkboek wordt getracht daar antwoord op te geven voor ondernemers in de veehouderij, de akkerbouw, de vollegrondsgroenteteelt en de fruitteelt. Tegelijkertijd worden suggesties gedaan hoe een agrariër met biodiversiteit kan ondernemen en wat dat voor het bedrijf betekent. Dit is een werkboek voor alle soorten en maten van grondgebonden bedrijven. Zowel gangbare, geïntegreerde als biologische bedrijven kunnen het gebruiken

Plants are continuously exposed to a wide variety of pathogens. However, all plant species are non-hosts for the majority of the potential plant pathogens. The genetic dissection of non-host resistance is hampered by the lack of segregating population from crosses between host and non-host species, since hardly any non-host is crossable with a host. We have studied the non-host resistance in Lactuca saligna (wild lettuce) to lettuce downy mildew ( Bremia lactucae ). L. saligna is one of the few examples of a non-host species that is crossable with a related host species, L. sativa (lettuce). Based on this interspecific cross, segregating populations have been developed for genetical analysis of the non-host resistance. To map the resistance, we have used two strategies in which we make use of DNA markers to genotype plants. As no accurate linkage map was available for lettuce, we started with the construction of a linkage map of L. saligna´L. sativa . In Chapter 2, the development of an integrated linkage map, based on two populations, is described. To acquire DNA markers, AFLP analyses have been performed on the F 2 populations of the crosses L. saligna CGN 5271 ´L. sativa Olof and L. saligna CGN 11341´L. sativa Norden. Based on these AFLP analyses the polymorphism rate between L. saligna and L. sativa is estimated to be 81%. A linkage map was constructed that comprises 12 SSRs and 476 AFLP markers over 854 cM in nine linkage groups (n=9). Since the markers are randomly spread over all chromosomes, we assume this map is an accurate representative of both parental genomes and very useful for Marker Assisted Selection.The first mapping strategy for downy mildew resistance is described in Chapter 3. In that study, we have performed a QTL analysis on 126 F 2 plants of a cross between the resistant L. saligna CGN 5271 and the susceptible L. sativa Olof. For this QTL analysis all 126 F 2 plants have been tested for resistance in four disease tests with two complex Bremia races (NL14 and NL16). The F 2 population showed a wide and continuous range of resistance levels from completely resistant to completely susceptible. Evidence is presented for a quantitative resistance against both Bremia races as well as for a race-specific resistance against Bremia race NL16 and not against NL14. These disease test data sets have been combined with DNA marker data of all 126 F 2 plants that had already been obtained for the construction of the linkage map. QTL mapping revealed a qualitative gene ( R39 ) explaining the race-specific resistance and three QTLs ( RBQ1 , RBQ2 and RBQ3 ) explaining the quantitative resistance. The qualitative gene R39 is a dominant gene that gives nearly complete resistance to race NL16 in L. saligna CGN 5271 and therefore it shows features similar to Dm genes (dominant race specific genes that give a complete resistance to d owny m ildew). The three QTLs explain 51% of the quantitative resistance against NL14, which indicates that probably not all QTLs have been detected in this F 2 population.In addition to this rather classical F 2 mapping strategy, we have performed an alternative mapping strategy based on the development and characterization of a set of Backcross Inbred Lines (BILs). These BILs are genetically nearly completely like L. sativa but contain a single chromosome substitution segment of L. saligna CGN 5271 (Chapter 4). Starting from an F 1 plant, BILs have been developed by four to five generations of backcrosses and one generation of selfing. All backcrosses from F 1 to BC 4 were made randomly without intentional selection. Marker Assisted Selection was started in the BC 4 generation. Finally, a set of 29 lines was obtained that covers 95% of the L. saligna genome, comprising 16 lines with a single homozygous introgression (BILs), one line with two homozygous introgressions, five lines with heterozygous single introgressions and seven lines with two or more heterozygous introgressions. Several chromosome regions showed severe distorted segregation in the F 2 population. Based on segregation ratios in backcross lines, we were able to explain distorted segregations of three chromosome regions observed in the F 2 population by genetic loci that are involved in pollen- or egg cell fitness.When seed of the first developed BILs was available, a disease test had been set up to test if the BILs, which carried QTLs as identified in the F 2 population, showed enhanced levels of quantitative resistance indeed. Nine BILs (or nearly-BILs) have been tested for resistance to Bremia race NL16. They covered together 31% of the L. saligna parental genome. Two resistance loci detected in the F 2 population ( R39 and RBQ3 ) have been confirmed in the disease test on the BILs. R39 is a dominant gene, which gives a complete resistance against Bremia race NL16. RBQ3 reduces the infection severity of the susceptible L. sativa by 49% ten days post inoculation. The quantitative effects from the resistance genes in these BILs were higher than expected from the F 2 mapping results. No conclusive comparisons of RBQ2 could have been made, as the introgression in the backcross line was not homozygous. RBQ1 has not been tested. Most exciting, the BIL method revealed a new resistance locus on Chromosome 8 with a 77% reduction on the infection severity compared to the susceptible control ten days post inoculation. We conclude that the BIL mapping method can reveal new QTLs unnoticed in the F 2 mapping method and it enables a quantification of the resistance gene effect in a L. sativa background.To extend our knowledge about the non-host resistance of L. saligna to Bremia , we have compared the genetics of non-host resistance to Bremia in L. saligna CGN 5271 with another accession L. saligna CGN 11341. The two accessions show a 39% AFLP polymorphism rate. We have analyzed the non-host resistance of L. saligna CGN 11341 by disease tests and DNA marker analyses on an F 2 and BC 1 population. Disease tests with Bremia races NL14 and NL16 showed a wide range of infection severity scores from resistant to susceptible to both races. The majority of plants had a similar resistance level to both Bremia races. These findings imply that the resistance of L. saligna is quantitatively expressed and is probably race non-specific. A few F 2 and BC 1 plants were completely resistant against Bremia race NL16 and rather susceptible to race NL14. QTL mapping revealed that a major resistance gene that was located on Chromosome 9 explains this race-specific resistance. This gene is designated R39b , as it may be different from R39 .No additional QTLs have been detected in this small F 2 population (n= 54). However, F 2 plants with L. saligna CGN 11341 alleles at loci of RBQ1 , RBQ2 , RBQ3 and RBQ4 mapped in L. saligna CGN 5271, were more resistant than F 2 plants with L. sativa alleles at these loci. In conclusion, we state that it is very likely that the same genes explain the resistances to Bremia in both L. saligna accessions. A backcross program for a set of Backcross Inbred Lines (BIL) that cover R39b and loci for putative QTLs, is in progress.In the last chapter of this thesis the basic results of the study have been discussed. We adduce that non-host resistance in L. saligna is not explained by accumulation of race-specific major resistance genes ( Dm genes) but by a resistance mechanism based on QTLs. Further, we have made a comparison for efficiency of four breeding methods to introgress the resistance genes from L. saligna . Based on this study, we conclude that twice as many resistance genes are introgressed when Marker Assisted Selection is used. Finally, several recommendations concerning research on non-host resistance and the applications of Backcross Inbred Lines have been suggested.

Plants are continuously exposed to a wide variety of pathogens. However, all plant species are non-hosts for the majority of the potential plant pathogens. The genetic dissection of non-host resistance is hampered by the lack of segregating population from crosses between host and non-host species, since hardly any non-host is crossable with a host. We have studied the non-host resistance in Lactuca saligna (wild lettuce) to lettuce downy mildew ( Bremia lactucae ). L. saligna is one of the few examples of a non-host species that is crossable with a related host species, L. sativa (lettuce). Based on this interspecific cross, segregating populations have been developed for genetical analysis of the non-host resistance. To map the resistance, we have used two strategies in which we make use of DNA markers to genotype plants. As no accurate linkage map was available for lettuce, we started with the construction of a linkage map of L. saligna´L. sativa . In Chapter 2, the development of an integrated linkage map, based on two populations, is described. To acquire DNA markers, AFLP analyses have been performed on the F 2 populations of the crosses L. saligna CGN 5271 ´L. sativa Olof and L. saligna CGN 11341´L. sativa Norden. Based on these AFLP analyses the polymorphism rate between L. saligna and L. sativa is estimated to be 81%. A linkage map was constructed that comprises 12 SSRs and 476 AFLP markers over 854 cM in nine linkage groups (n=9). Since the markers are randomly spread over all chromosomes, we assume this map is an accurate representative of both parental genomes and very useful for Marker Assisted Selection.The first mapping strategy for downy mildew resistance is described in Chapter 3. In that study, we have performed a QTL analysis on 126 F 2 plants of a cross between the resistant L. saligna CGN 5271 and the susceptible L. sativa Olof. For this QTL analysis all 126 F 2 plants have been tested for resistance in four disease tests with two complex Bremia races (NL14 and NL16). The F 2 population showed a wide and continuous range of resistance levels from completely resistant to completely susceptible. Evidence is presented for a quantitative resistance against both Bremia races as well as for a race-specific resistance against Bremia race NL16 and not against NL14. These disease test data sets have been combined with DNA marker data of all 126 F 2 plants that had already been obtained for the construction of the linkage map. QTL mapping revealed a qualitative gene ( R39 ) explaining the race-specific resistance and three QTLs ( RBQ1 , RBQ2 and RBQ3 ) explaining the quantitative resistance. The qualitative gene R39 is a dominant gene that gives nearly complete resistance to race NL16 in L. saligna CGN 5271 and therefore it shows features similar to Dm genes (dominant race specific genes that give a complete resistance to d owny m ildew). The three QTLs explain 51% of the quantitative resistance against NL14, which indicates that probably not all QTLs have been detected in this F 2 population.In addition to this rather classical F 2 mapping strategy, we have performed an alternative mapping strategy based on the development and characterization of a set of Backcross Inbred Lines (BILs). These BILs are genetically nearly completely like L. sativa but contain a single chromosome substitution segment of L. saligna CGN 5271 (Chapter 4). Starting from an F 1 plant, BILs have been developed by four to five generations of backcrosses and one generation of selfing. All backcrosses from F 1 to BC 4 were made randomly without intentional selection. Marker Assisted Selection was started in the BC 4 generation. Finally, a set of 29 lines was obtained that covers 95% of the L. saligna genome, comprising 16 lines with a single homozygous introgression (BILs), one line with two homozygous introgressions, five lines with heterozygous single introgressions and seven lines with two or more heterozygous introgressions. Several chromosome regions showed severe distorted segregation in the F 2 population. Based on segregation ratios in backcross lines, we were able to explain distorted segregations of three chromosome regions observed in the F 2 population by genetic loci that are involved in pollen- or egg cell fitness.When seed of the first developed BILs was available, a disease test had been set up to test if the BILs, which carried QTLs as identified in the F 2 population, showed enhanced levels of quantitative resistance indeed. Nine BILs (or nearly-BILs) have been tested for resistance to Bremia race NL16. They covered together 31% of the L. saligna parental genome. Two resistance loci detected in the F 2 population ( R39 and RBQ3 ) have been confirmed in the disease test on the BILs. R39 is a dominant gene, which gives a complete resistance against Bremia race NL16. RBQ3 reduces the infection severity of the susceptible L. sativa by 49% ten days post inoculation. The quantitative effects from the resistance genes in these BILs were higher than expected from the F 2 mapping results. No conclusive comparisons of RBQ2 could have been made, as the introgression in the backcross line was not homozygous. RBQ1 has not been tested. Most exciting, the BIL method revealed a new resistance locus on Chromosome 8 with a 77% reduction on the infection severity compared to the susceptible control ten days post inoculation. We conclude that the BIL mapping method can reveal new QTLs unnoticed in the F 2 mapping method and it enables a quantification of the resistance gene effect in a L. sativa background.To extend our knowledge about the non-host resistance of L. saligna to Bremia , we have compared the genetics of non-host resistance to Bremia in L. saligna CGN 5271 with another accession L. saligna CGN 11341. The two accessions show a 39% AFLP polymorphism rate. We have analyzed the non-host resistance of L. saligna CGN 11341 by disease tests and DNA marker analyses on an F 2 and BC 1 population. Disease tests with Bremia races NL14 and NL16 showed a wide range of infection severity scores from resistant to susceptible to both races. The majority of plants had a similar resistance level to both Bremia races. These findings imply that the resistance of L. saligna is quantitatively expressed and is probably race non-specific. A few F 2 and BC 1 plants were completely resistant against Bremia race NL16 and rather susceptible to race NL14. QTL mapping revealed that a major resistance gene that was located on Chromosome 9 explains this race-specific resistance. This gene is designated R39b , as it may be different from R39 .No additional QTLs have been detected in this small F 2 population (n= 54). However, F 2 plants with L. saligna CGN 11341 alleles at loci of RBQ1 , RBQ2 , RBQ3 and RBQ4 mapped in L. saligna CGN 5271, were more resistant than F 2 plants with L. sativa alleles at these loci. In conclusion, we state that it is very likely that the same genes explain the resistances to Bremia in both L. saligna accessions. A backcross program for a set of Backcross Inbred Lines (BIL) that cover R39b and loci for putative QTLs, is in progress.In the last chapter of this thesis the basic results of the study have been discussed. We adduce that non-host resistance in L. saligna is not explained by accumulation of race-specific major resistance genes ( Dm genes) but by a resistance mechanism based on QTLs. Further, we have made a comparison for efficiency of four breeding methods to introgress the resistance genes from L. saligna . Based on this study, we conclude that twice as many resistance genes are introgressed when Marker Assisted Selection is used. Finally, several recommendations concerning research on non-host resistance and the applications of Backcross Inbred Lines have been suggested.

Kritische beschouwing over het denkbeeld van J.E. Vanderplank ten aanzien van horizentale en verticale resistentie bij planten

Kritische beschouwing over het denkbeeld van J.E. Vanderplank ten aanzien van horizentale en verticale resistentie bij planten

Key-words: bacterial blight, biological control, biodiversity, endophytic bacteria, L-form, pea, PDRl retrotransposon, Pisum sativum, Pisum abyssinicum, Pseudomonas syringae pv. pisi, race specific resistance, race non-specific resistance, Spanish landraces.Pea bacterial blight (Pseudomonas syringae pv. pisi) occurs worldwide and can cause severe damage under cool and wet conditions particularly at the seedling stage in wintersown crops. Seven Ps. syr. pv. pisi races are currently recognized. There are no resistant cultivars to race 6, which is becoming increasingly important. Current disease control measures include disease avoidance through seed testing and the deployntent of resistant cultivars with race specific resistance gene(s). In the present study two novel control measures were investigated with the potential for integration to give a durable and sustainable disease control. The first was breeding for resistance based on race non-specific resistance present in Pisum abyssinicum, which confers resistance to all races, including race 6. Its mode of inheritance was investigated through a crossing programme with Pisum sativum cultivars. Resistance was controlled by a major recessive gene and a number of modifiers. Progenies of crosses between resistant F5 populations and commercial cultivars are now available. Molecular markers for race non-specific resistance based on a pea retrotransposon marker system were developed. It is suggested that the combination of race specific and race non-specific resistance provides the optimal genetic background for the maximum expression of resistance to all races of the pathogen in all plant parts and under field conditions. The second measure was biological control by endophytic bacteria. Studies on the ecology of endophytic bacteria in pea and identification of efficient indigenous colonizers for potential application in biocontrol have been made. Endophytic population levels were in the range 10 3 -10 6 CFU/g fresh tissue in roots and stems. There was a predominance of Gram-negative bacteria, particularly Pseudomonas sp. and Pantoea agglomerans. Arthrobacter sp. and Curtobacterium sp. were the main Gram-positive bacteria. Factors such as soil type, plant genotype and crop growth stage may significantly influence the diversity and population levels of endophytic bacteria. Future research should focus on the combination and testing of elite breeding lines with selections of disease suppressive endophytic isolates under a variety of field conditions in order to obtain an efficient and durable performance in commercial agriculture.

Key-words: bacterial blight, biological control, biodiversity, endophytic bacteria, L-form, pea, PDRl retrotransposon, Pisum sativum, Pisum abyssinicum, Pseudomonas syringae pv. pisi, race specific resistance, race non-specific resistance, Spanish landraces.Pea bacterial blight (Pseudomonas syringae pv. pisi) occurs worldwide and can cause severe damage under cool and wet conditions particularly at the seedling stage in wintersown crops. Seven Ps. syr. pv. pisi races are currently recognized. There are no resistant cultivars to race 6, which is becoming increasingly important. Current disease control measures include disease avoidance through seed testing and the deployntent of resistant cultivars with race specific resistance gene(s). In the present study two novel control measures were investigated with the potential for integration to give a durable and sustainable disease control. The first was breeding for resistance based on race non-specific resistance present in Pisum abyssinicum, which confers resistance to all races, including race 6. Its mode of inheritance was investigated through a crossing programme with Pisum sativum cultivars. Resistance was controlled by a major recessive gene and a number of modifiers. Progenies of crosses between resistant F5 populations and commercial cultivars are now available. Molecular markers for race non-specific resistance based on a pea retrotransposon marker system were developed. It is suggested that the combination of race specific and race non-specific resistance provides the optimal genetic background for the maximum expression of resistance to all races of the pathogen in all plant parts and under field conditions. The second measure was biological control by endophytic bacteria. Studies on the ecology of endophytic bacteria in pea and identification of efficient indigenous colonizers for potential application in biocontrol have been made. Endophytic population levels were in the range 10 3 -10 6 CFU/g fresh tissue in roots and stems. There was a predominance of Gram-negative bacteria, particularly Pseudomonas sp. and Pantoea agglomerans. Arthrobacter sp. and Curtobacterium sp. were the main Gram-positive bacteria. Factors such as soil type, plant genotype and crop growth stage may significantly influence the diversity and population levels of endophytic bacteria. Future research should focus on the combination and testing of elite breeding lines with selections of disease suppressive endophytic isolates under a variety of field conditions in order to obtain an efficient and durable performance in commercial agriculture.

Het toetsen van verschillen in gevoeligheid van komkommerrassen voor echte meeldauw vraagt veel tijd en ruimte. Bij het in dit verslag beschreven onderzoek is gezocht naar een methode OM dergelijke vergelijkingen te vereenvoudigen. In een proef met 17 rassen, waarvan een deel partieel resistent, is van elk ras een aantal planten op één echt blad getopt en een aantal is, zoals tot nu toe gebruikelijk bij toetsen op echte meeldauw, normaal opgeleid en pas op 10 é 11 bladeren getopt. Alle planten zijn op het moment dat ze 17 dagen oud waren en het eerste echte blad een diameter van circa 10 cm had, geïnoculeerd met een sporensuspensie met 1.000 sporen per ml. Er zijn waarnemingen gedaan ten aanzien van de incubatietijd en het aantal meeldauwvlekken op 50 cm2 van het geïnoculeerde blad. Daarnaast zijn de aantastingen op verschillende momenten gewaardeerd. Gebleken is dat de waardering van primaire aantasting op getopte en niet getopte planten nagenoeg gelijk is zodat voor het aangeven van verschillen tussen rassen van een primaire aantasting volstaan kan worden met getopte planten. Er bleek ook een zeer nauw verband te bestaan tussen de primaire aantasting op de getopte planten 16 dagen na inoculatie en de primaire + secundaire aantasting op de niet getopte planten 28 dagen na inoculatie. Hieruit kan geconcludeerd worden dat bij toetsen van gevoeligheidsverschillen tussen komkommerrassen gebruik gemaakt kan worden van planten die direct na inoculatie op één echt blad worden getopt. De primaire aantasting op het geïnoculeerde blad geeft een goed beeld van de gevoeligheidsverschillen tussen de rassen. Met deze toetsmethode kan grote tijd- en ruimtewinst worden geboekt.

Het toetsen van verschillen in gevoeligheid van komkommerrassen voor echte meeldauw vraagt veel tijd en ruimte. Bij het in dit verslag beschreven onderzoek is gezocht naar een methode OM dergelijke vergelijkingen te vereenvoudigen. In een proef met 17 rassen, waarvan een deel partieel resistent, is van elk ras een aantal planten op één echt blad getopt en een aantal is, zoals tot nu toe gebruikelijk bij toetsen op echte meeldauw, normaal opgeleid en pas op 10 é 11 bladeren getopt. Alle planten zijn op het moment dat ze 17 dagen oud waren en het eerste echte blad een diameter van circa 10 cm had, geïnoculeerd met een sporensuspensie met 1.000 sporen per ml. Er zijn waarnemingen gedaan ten aanzien van de incubatietijd en het aantal meeldauwvlekken op 50 cm2 van het geïnoculeerde blad. Daarnaast zijn de aantastingen op verschillende momenten gewaardeerd. Gebleken is dat de waardering van primaire aantasting op getopte en niet getopte planten nagenoeg gelijk is zodat voor het aangeven van verschillen tussen rassen van een primaire aantasting volstaan kan worden met getopte planten. Er bleek ook een zeer nauw verband te bestaan tussen de primaire aantasting op de getopte planten 16 dagen na inoculatie en de primaire + secundaire aantasting op de niet getopte planten 28 dagen na inoculatie. Hieruit kan geconcludeerd worden dat bij toetsen van gevoeligheidsverschillen tussen komkommerrassen gebruik gemaakt kan worden van planten die direct na inoculatie op één echt blad worden getopt. De primaire aantasting op het geïnoculeerde blad geeft een goed beeld van de gevoeligheidsverschillen tussen de rassen. Met deze toetsmethode kan grote tijd- en ruimtewinst worden geboekt.

Kritische beschouwing over het denkbeeld van J.E. Vanderplank ten aanzien van horizentale en verticale resistentie bij planten

In het kader van het onderzoeksprogramma 'Biologische productiesystemen in de akkerbouw en vollegrondsgroenteteelt' is de afgelopen vier jaar onderzoek gedaan naar de mogelijkheden die veredeling kan bieden om het wortelstelsel van uien te verbeteren. Dit lijkt mogelijk door specifieke soortkruisingen met stengelui (Japanse bladui) en Allium roylei, met als bijkomend voordeel dat naast de selectie op een beter type wortelstelsel ook geselecteerd kan worden op resistentie tegen de bladschimmels valse meeldauw en bladvlekkenziekte. Een opvallend onderzoeksresultaat was het effect van arbusculaire mycorrhiza (AM) op het verbeteren van het wortelstelsel en de productiviteit van stengelui, en mogelijk ook ui