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.

Field experiments were carried out (2001-2003) to evaluate the efficacy of combining host resistance with reduced rates and frequencies of the residual contact fungicide fluazinam to control foliar potato late blight (Phytophthora infestans). Potato cultivars (cvs.) and advanced breeding lines (ABL) developed in four states were used in combination with fluazinam applications at 0, 33%, 50%, 66% and 100% of the manufacturer recommended application rate (MRAR) applied at 5, 7, 10 or 14 day application intervals. Values of relative area under disease progress curve (RAUDPC) in the untreated plots demonstrated that cvs. and ABL were significantly different in susceptibility to late blight. The cv. Jacqueline Lee was used as a resistant reference and the cvs. Snowden and FL1879 as susceptible. Accordingly, the ABL MSJ317-1 and MSJ461-1 were classified as resistant and the ABL W1386, W1201, MN19515, MN15620, MN19157, MN98650-8, ND2470-27, ND5822C-7 as susceptible. The cvs./ABL Dakota Pearl, Dakota Rose, MN19350 and W1355-1 were classified as susceptible in 2001 and as moderately susceptible in 2002. Application of the full rate of fluazinam at 5 or 7 day intervals resulted in effective control in all cvs./ABL except the cv. Dakota Pearl at 7 day application interval in 2001; at 10 or 14 day intervals it provided effective control in some cv./ABL and either partial or ineffective control in others. The 50% or 66% MRAR of fluazinam applied at 5 or 7 days provided effective blight control in resistant and moderately susceptible cvs./ABL and partial control in susceptible cvs./ABL. Fluazinam applied at 33% MRAR (in 2001 trials), at any application interval, provided effective blight control in the resistant cvs. and was either partially effective or ineffective in the susceptible cvs.. The study demonstrates that potato cvs./ABL with reduced susceptibility to late blight can be managed with reduced fungicide rates and longer application intervals, thus offering a less expensive option for potato late blight control.

Several greenhouse inoculation methods are available to evaluate soybean (Glycine max (L.) Merr.) for resistance to Sclerotinia sclerotiorum (Lib.) de Bary. Most of these methods are labor intensive and often produce inconsistent results among the tests. The objective of this research was to develop a low-cost and high-efficiency greenhouse inoculation method that can generate a consistent result. We developed a spray-mycelium method in which mycelia were cultured in liquid potato dextrose broth and homogenized before spraying on the soybean leaves. We also developed an inoculation method (the "drop-mycelium" method) in which a drop of homogenized mycelium suspension was dropped on the tips of main stems. Inoculated plants were incubated in a greenhouse chamber with 60 to 80% relative humidity. Plant mortality and area under the wilt progress curve (AUWPC) were used to measure disease severity daily from 3 to 14 days after inoculation (DAI). Eighteen soybean genotypes, including partially resistant line NKS19-90 and susceptible line Resnik, were employed in this study. The spray-mycelium method and the drop-mycelium method were compared with the cut-petiole method in the greenhouse. The three experiments were a randomized complete block design. Twenty-four plants per genotype in each experiment were inoculated at V3 growth stage in the greenhouse. Significant differences (P < 0.05) in disease ratings of plant mortality and AUWPC to Sclerotinia stem rot were found among 18 tested genotypes. The results obtained with the spray-mycelium and drop-mycelium inoculation methods were significantly (R > 0.73, P < 0.01) correlated with the results obtained with the cut-petiole inoculation method for both of the plant mortality and AUWPC. Compared with the cut-petiole method, the spray-mycelium and the drop-mycelium methods used less inoculation time and are less expensive in terms of materials. Both of these new methods are low cost, efficient, and reliable and they can be valuable for large-scale evaluation of germ plasm and breeding lines for resistance to Sclerotinia stem rot in a greenhouse or other similar facilities.

Die Kartoffel zählt neben Getreide zu den zwei wichtigsten Grundnahrungsmittel in Europa. Die Bedeutung der Kartoffelkrankheit Silberschorf, hervorgerufen durch Helminthosporium solani (Dur. et Mont.), hat in den letzten 10 Jahren stark zugenommen. In Extremfällen kann es durch die Krankheit zu Verlusten von bis zu 80% der geernteten Ware wegen Qualitätsverlust kommen. Die Bekämpfung des Erregers stellt sich als äußerst schwierig dar. Weder pflanzenbauliche Maßnahmen noch der Einsatz von Fungiziden ist aufgrund von Resistenzen des Pilzes gegen die verfügbaren Wirkstoffe erfolgreich. Der vielversprechendste Weg zur Bekämpfung des Silberschorfs ist die Züchtung resistenter Kartoffelsorten. Erstes Ziel der Doktorarbeit war es, die Anfälligkeit verschiedener Kartoffelsorten auf H. solani zu ermitteln. Dabei sollte der Einfluss äußerer Faktoren wie Klima, Witterung oder Boden berücksichtigt werden. In dieser Arbeit wurde ein Standartsortiment von 12 Kartoffelsorten mit definierter Anfälligkeit erstellt. Es konnten keine resistenten Sorten gefunden werden. Alle Kartoffeln zeigten eine sortenabhängige Anfälligkeit gegen Silberschorf. Die Anfälligkeit variierte von stark anfälligen bis wenig anfälligen Sorten. Dabei hatten äußere Faktoren kaum einen Einfluss. Die Unterschiede in der Anfälligkeit blieben sortenspezifisch an den verschiedenen Standorten, zwischen den verschiedenen Jahren, im Gewächshaus sowie im Lager bestehen.Zweites Ziel der Arbeit war die Untersuchung der Ursachen der unterschiedlichen Anfälligkeit der Sorten (Resistenzfaktoren). Der in der Literatur beschriebene Zusammenhang zwischen Anfälligkeit und Schalendicke konnte nicht bestätigt werden. Auch die Schalenbeschaffenheit scheint keinen Einfluss zu haben. Ein weiterer untersuchter Parameter ist der Einfluss phenolischer Substanzen in der Kartoffelschale. Verschiedene Methoden zur Extraktion von Phytoalexinen sowie Ligninen und dessen Vorstufen aus Kartoffelschalen wurden getestet für eine quantitative photometrische Messung und qualitative HPLC-Analysen. Ein Einfluss phenolischer Komponenten auf die Anfälligkeit konnte nicht festgestellt werden. In wieweit dies auf die für Kartoffelschalen möglicherweise ungeeigneten Extraktionsmethoden zurückzuführen ist, ist unklar. Weiterhin wurde gezeigt, dass verschiedene Böden eine suppressive Wirkung auf die Keimung der H. solani Konidien haben. Es wurde ermittelt, dass bestimmte Kartoffelsorten diese Keimsuppression durch einen noch unbekannten Mechanismus teilweise wieder aufheben können. Die Keiminduktion stand in direkter, signifikanter Beziehung zu den Anfälligkeiten der Sorten. Zur Erforschung der Ursachen wurden Zuckeranalysen durchgeführt. Es konnte keine Beziehung zwischen Zuckergehalt und Keimförderung gefunden werden.Drittes Ziel der Arbeit war die Erforschung des Übertragungswegs des Pilzes von Mutter- auf Tochterknollen im Boden. Es wurde gezeigt, dass der passive Transport der Konidien im Boden mittels Wasser nur eine untergeordnete Rolle zu spielen scheint. Eine direkte Übertragung des Pilzes durch aktives Mitwachsen in den Stolonen wurde mittels PCR-Analysen nachgewiesen. In Stolonenproben befallener Knollen verschiedener Sorten konnte H. solani gefunden werden. Die direkte Übertragung über Stolonen scheint somit nicht nur möglich zu sein, sondern auch eine wichtige Rolle zu spielen. Eine systemische Ausbreitung des Pilzes wurde durch negative Ergebnisse in PCR-Untersuchungen der Leitbündel befallener Knollen ausgeschlossen. | The potato is, together with cereals, one of the two most important stable food in human nutrition because of its precious ingredients. Silver scurf, caused by Helminthosporium solani (Dur. & Mont.) represents an increasing problem in potato cultivation. In extreme cases, the yield loss because of quality loss can be more then 80 %. The control of the disease is exceedingly difficult. Neither plant cultivating arrangements nor adequate fungicide because of resistances of the fungus are available. The most promising solution is the breeding of resistant potato cultivars. The first aim of this project was to work out the susceptibilities of different potato cultivars against silver scurf. The influence of outer factors like weather or soil conditions should be considered. In this work a standard assortment of potato with a defined susceptibility could be established. No resistant genotype was found. The susceptibility varied from very susceptible to slightly susceptible cultivars. Outer factor hardly showed any effect and could be excluded to the greatest possible extent. The differences in susceptibility of the cultivars were analogue in different location, during several years, in greenhouse and during storage.The second aim was the detection of the causes of the differences in susceptibility (resistance factors). A relation between peel thickness and susceptibility could not be approved. As well the peel consistency seems to have no influence. Another investigated parameter was the amount of phenolic compounds within the potato peel. Several methods for extraction of phytoalexines and Lignin with its pre-stages in potato peel where proved for a quantitative photometric measurement and qualitative test via HPLC analyses. A influence of phenolic compounds on the susceptibility of the cultivars could no be examined. It is unclear how far these results are dependent on the unsuitable extraction methods for potato peels. It was also found, that different soils had a suppressive impact on the germination of the H. solani conidias. Furthermore it was discovered, that several potato cultivars can somehow increase the amount of germinated conidias again. This germination induction was closely related to the susceptibility of the cultivars. To investigate the causes, sugar analyses were carried out. There was no relation between sugar amount and germination induction. The third aim was the investigation of the devolution of the fungus from mother to daughter tubers in soil. It was shown that the transportation through soil just plays a secondary role in conidia devolution. A direct devolution through stolones was examined via PCR trials. In stolone samples H. solani was detected. This leads to the conclusion that direct devolution is not only possible but also plays an important role. A systemical devolution was excluded with negative results of PCR trials of the vascular bundle of infected tubers.

In this thesis research is described aiming at alleviation of the perceived limitations in the standard protocol which encompasses: mapping a trait, followed by marker saturation, genetic resolution, and finally BAC landing and walking to span the physical distance between the markers.In Chapter 2 proof of concept is presented that the level of marker saturation offered by the current version of the ultra dense map of potato is sufficient for map based cloning efforts. This is demonstrated by the assembly of a contig enclosing the Sen1-4 gene involved in wart disease resistance.Current linkage maps generated with AFLP show the unpleasant feature of centromeric clustering of genomic markers due to centromeric repression of recombination. Methods to target markers in the heterochromatic parts of the genome and to avoid the centromeric euchromating depend on e.g. methylation. AFLP markers based on the methylation sensitive enzyme Pst I show a more uniform coverage of the linkage map. Another method to target the gene rich parts of the genome is the use of cDNA as template for marker development. In the past RFLP probes were derived from Pst I digested libraries or cDNA clones. In the era of PCR based marker techniques it is evident that cDNA-AFLP might be a useful method; not only for transcript profiling but also for marker development. Furthermore this may also help to circumvent the time consuming BAC walking, when gene landing in stead of BAC landing might be an option. To enable gene landing, a marker has to be derived from the gene itself. When cDNA-AFLP patterns are generated from a series of offspring genotypes from a mapping population, then the polymorphisms in these cDNA-AFLP fingerprints should serve as a genetic marker of a specific chromosomal position. In Chapter 3 markers are generated in a mapping population using the cDNA-AFLP technique to evaluate and test the applicability of these markers.AFLP fingerprinting, both with genomic DNA as well as cDNA as the source material for template production, is a random technique. It does not allow specific targeting of gene rich regions in general or certain DNA sequences in particular. However, at this moment novel combinations of selective AFLP primers and specific primers allow us to generate fingerprints with amplification products that represent sequence tags of specific classes of genes. One of these AFLP-derived gene-family-specific fingerprinting techniques is called NBS profiling. This enables the specific amplification of parts of resistance genes or resistance gene analogs. In Chapter 4 NBS profiling is performed on templates prepared from DNA isolated from a mapping population to evaluate the potential to genetically map resistance gene clusters. In addition, cDNA can be used to evaluate the expression of R-genes between tissues in combination with NBS-profiling.AFLP and anchor related markers are rather laborious and expensive because several enzymes and amplification steps are needed before a product is ready for separation. This has to be performed on a polyacrylamide gel based system to create the resolution that is needed for accurate separation of the fragments. This time consuming and expensive protocol make anchor related techniques not suitable for recombinant screenings or marker assisted selection (MAS). Moreover, the required equipment is often unavailable to breeding companies. Therefore, a protocol for the efficient conversion of AFLP markers into simple PCR markers was developed (Chapter 5).Finally in chapter 6 the potential of the different methods to improve the efficiency for gene mapping and gene cloning is discussed.