Identification of resistance sources and characterization of resistance factors in Brassica species to Verticillium longisporum

Abstract

The increasing area of oilseed rape cultivation particularly in the northern European countries has significantly promoted Verticillium longisporum, the causal agent of "Verticillium wilt" of Brassica crops, and thus has rendered this disease a major challenge for current resistance breeding efforts. One of the most promising means of controlling Verticillium within the scope of an integrated pest management in addition to the implementation of cultural practices is the use of resistant cultivars. Until now, for both winter and spring type oilseed rape, breeding for resistance has been severely hampered by the absence of sufficient resistance in commercially available breeding material. With these considerations, the chief objectives of this dissertation were to identify new sources of resistance and to characterize resistance factors in oilseed rape genotypes as well as in related Brassica forms with the long-term goal of improving the resistance of winter oilseed rape against V. longisporum. One major part of this work is concerned with the screening of different Brassica species, including B. napus, resynthesized oilseed rape forms and the progenitor species B. oleracea and B. rapa (syn. campestris) both in greenhouse and field studies. Due to the lack of correlation between greenhouse and field results, it is concluded that the eligibility of the screening assay as an isolated test method is equivocal. Thus, in order to identify genotypes that are undoubtedly resistant not only under standardized but also under field conditions, a combination of upstream greenhouse screenings and ensuing resistance tests in the field at several locations is indispensable. A further objective of this work was to identify mechanisms contributing to the overall resistance phenotype of Brassica varieties to V. longisporum and potentially useful for oilseed rape breeding programmes. In principle, two different approaches were pursued throughout this project: In the first instance, potential levels of resistance were identified by investigating the interaction of B. napus with a virulent, host-specific isolate of V. longisporum in comparison to the interaction with a host-heterologous strain of V. dahliae. In the first approach, the differential interactions of V. longisporum and V. dahliae on the root surface and in the root and shoot vascular system of B. napus were studied by confocal laser scanning microscopy (CLSM), using GFP tagging and conventional fluorescence dyes. This study provided novel information about the early stages of infection and colonization in the host and non-host-pathogen-interaction. Although V. dahliae was infrequently able to penetrate roots of B. napus, it failed to spread further into the shoot, as shown with real-time PCR. Furthermore, screening material with different levels of resistance to V. longisporum was used to differentially characterize relevant resistance factors. As a result, mechanisms were found which become operative after the pathogen has entered the plant, such as the build up of mechanical barriers like tyloses or vascular gels, as well as the reinforcement of constitutive barriers through the deposition of cell wall-bound phenols and lignin. Furthermore, the accumulation of soluble phenolics was observed. Although similar responses occurred in vascular tissues of both resistant and susceptible plants, they occurred with a higher intensity in the resistant B. napus and B. oleracea accessions. Thus, from these studies, differences in responses between the susceptible and the resistant interaction can be regarded as being quantitative rather than qualitative in nature. In both resistant Brassica genotypes the most active defence occurs in the hypocotyl tissue and seems to be based on similar if not the same mechanisms. This clearly corroborates the hypothesis that resistance traits in B. napus have been introgressed from B. oleracea.