Oilseed rape (Brassica napus) is one of the major oilseed crops in the world but is vulnerable to attack by many pathogens and insect pests. In addition to the host plant genotype, micro-organisms present in the rhizosphere and within plant tissues affect the susceptibility to plant pathogens. While rapid progress has been achieved concerning the concept of plant resistance genes, information on the role of the microbial community in plant protection is less apparent. We have studied the endophytic bacterial populations present in different tissues of oilseed rape and also analysed several cultivars (Express, Libraska, Maluka and Westar), which differ in their susceptibility to the wilt pathogen Verticillium longisporum. The population diversity was studied using agar plating assay, fatty acid methyl ester analysis and functional characterisation of isolated strains. Our work shows that already in the seeds there exists diversity in populations as well as in the total microbial load between two of the four tested cultivars. About 50% of the strains isolated from cultivars Express and Libraska showed moderate to strong direct inhibition of V. longisporum. The diversity of the endophytic flora isolated from oilseed rape and its implications in crop protection are discussed.

The enzyme myrosinase (EC 3.2.3.1) degrades the secondary compounds glucosinolates upon wounding and serves as a defense to generalist pests in Capparales. Certain myrosinases are present in complexes together with other proteins such as myrosinase-binding proteins (MBP) in extracts of oilseed rape (Brassica napus) seeds. Immunhistochemical analysis of wild-type seeds showed that MBPs were present in most cells but not in the myrosin cells, indicating that the complex formation observed in extracts is initiated upon tissue disruption. To study the role of MBP in complex formation and defense, oilseed rape antisense plants lacking the seed MBPs were produced. Western blotting and immunohistochemical staining confirmed depletion of MBP in the transgenic seeds. The exclusive expression of myrosinase in idioblasts (myrosin cells) of the seed was not affected by the down-regulation of MBP. Using size-exclusion chromatography, we have shown that myrosinases with subunit molecular masses of 62 to 70 kD were present as free dimers from the antisense seed extract, whereas in the wild type, they formed complexes. In accordance with this, MBPs are necessary for myrosinase complex formation of the 62-to 70-kD myrosinases. The product formed from sinalbin hydrolysis by myrosinase was the same whether MBP was present or not. The performance of a common beetle generalist (Tenebrio molitor) fed with seeds, herbivory by flea beetles (Phyllotreta undulata) on cotyledons, or growth rate of the Brassica fungal pathogens Alternaria brassicae or Lepthosphaeria maculans in the presence of seed extracts were not affected by the down-regulation of MBP, leaving the physiological function of this protein family open.Specific ␤-thioglucosidases known as myrosinases (EC 3.2.3.1) are responsible for the degradation of glucosinolates, secondary metabolites found mainly in the members of the Brassicacea family (Chew, 1988; James and Rossiter, 1991; Louda and Mole, 1991). The myrosinases and glucosinolates are, to a large extent, preformed and most probably require disruption of the plant tissue to become activated. Myrosinase hydrolyses glucosinolates by releasing the Glc moiety. The remaining aglycone spontaneously rearranges into one of several toxic products, such as isothiocyanates, thiocyanates, and nitriles (for review, see Bones and Rossiter, 1996; Rask et al., 2000). The outcome of the degradation depends on the type of glucosinolate being degraded, the local milieu, and additional proteins such as the epithiospecifier protein (Chew, 1988; Louda and Mole, 1991; Lambrix et al., 2001).Several different myrosinase isoenzymes have been characterized in seeds, seedlings, and vegetative tissues of oilseed rape (Brassica napus; Lenman et al., 1993). More than 20 myrosinase genes seem to be present in oilseed rape (Xue et al., 1992), but few of these genes or cDNAs have been cloned as yet. Myrosinases expressed in seeds can be divided into three different subfamilies, denoted MA, MB, and MC. The three-dimensional structure of one myrosina...

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summary Leptosphaeria maculans causes blackleg disease, and resistance to this fungal pathogen is an important trait in the breeding of oilseed rape. A better comprehension of the role of the myrosinase-glucosinolate system in this context is of great value. The present study is the first to address effects on multiple components of this complex system, including concentrations of individual glucosinolates, product formation, myrosinase isoform distribution and activity, and levels of myrosinase binding proteins during the infection process. One resistant B. napus cultivar (Maluka) and one susceptible cultivar (Westar) were compared in the investigation. Our results show that the two cultivars had the same histological distribution, isoform expression, and activity of the myrosinase enzymes. The glucosinolate levels were also similar, with the exception of glucobrassicin and neoglucobrassicin, which were significantly lower in the resistant cultivar at 11 days post-infection. Growth of the fungus on the plant tissues did not alter glucosinolate levels, suggesting that L. maculans does not degrade these compounds. When the plants were starved of sulphur, and thereby depleted of glucosinolates, no increased susceptibility was observed. Hence, we suggest that the myrosinase-glucosinolate system does not determine the outcome of the interaction between B. napus and L. maculans.