Improvement of resistance in oilseed rape (Brassica napus L.) to stem rot induced by Sclerotinia sclerotiorum

Aim of the project

Sclerotinia sclerotioum is a major pathogen on oilseed rape. Infestation levels of up to 40% which have been reported from Schleswig-Holstein and Mecklenburg-Vorpommern (Dunker & Tiedemann, 2004) may cause yield losses of up to 50% (Pope et al., 1989), as a result of a reduced thousand seed weight or an early shattering of pods.
The set of winter and summer oilseed rape cultivars registered in Germany comprises no effective resistance to stem rot (Sclerotinia sclerotiorum). The aim of this project, which is conducted in collaboration with GFP and oilseed rape breeding companies, is the provision of suitable screening methods for a systematic search for potential resistance sources in the diverse gene pools related to Brassica napus. Valuable material will be made available for breeding companies involved in the project. A second issue of the project is to provide improved and reliable assessment methods suitable for the official classification of cultivars.

Field screening of B. napus genotypes

In 2007/2008, a field experiment was conducted in which the plants were inoculated by spraying with a mycelium suspension. Despite the fact, that the chosen inoculation method was not as successful as assumed, we were able to discern between the 24 cultivars tested due to the natural disease pressure in the field (up to 30% infected plants). In 2008/2009, a field experiment was designed, in which 28 cultivars, which were provided by the participating breeders, were screened. This experiment was conducted on three locations in Germany and the disease pressure was increased by spreading out sclerotia. Unfortunately, due to weather conditions during flowering the inoculation was again not successful and thus the experiment will be repeated in the same way in 2009/2010.

Root inoculation experiments

S. sclerotiorum have been reported in recent years from several fields in Germany and France and represent a novel threat by this pathogen. We collected isolates from infected roots and inoculated oilseed rape roots in the greenhouse. Several inoculation methods were used and an experiment was conducted in which the influence of different temperatures was investigated. Placement of agar plugs overgrown with mycelium in direct contact to the primary root turned out to be an appropriate inoculation method. We will now investigate, whether root-derived isolates of S. sclerotiorum behave differently from shoot-derived isolates. Moreover, we are interested to compare responses of genotypes to shoot and root inoculations, in order to test whether there exists a plant organ specific resistance or susceptibility.

Screening for resistance in the laboratory and greenhouse

A laboratory screening is needed to enable an elevated through-put of genotypes to be tested. The lab assay consists of a test for sensitivity of plant tissue to oxalic acid, a crucial pathogenicity factor of S. sclerotiorum. Preliminary data from this test fit very well with data from the screening in the field (2007/2008). Further adjustments and testing is currently ongoing to verify the reliability and efficiency of this lab assay.
The greenhouse screening is performed with flowering plants (growth stage 63 to 65) which are inoculated by placing an agar plug with mycelium into the axil of a leaf or side shoot. Longitudinal lesion growth is measured periodically, allowing for conclusions on the tissue resistance of the genotype tested. Thus a broad range of different plants from various gene pools will be tested to identify resistance sources. In addition, results will be related to inoculation experiments with ascospores artificially raised in the lab.

Transformation of B. napus

In order to adjust and verify the above assessment methods we also use B. napus plants with transgenic resistance to S. sclerotiorum. Two gene constructs were received from Dr. Andreas Walz (University Hohenheim) as expression constructs under the control of the 35S promoter of the cauliflower mosaic virus. Both genes carry a kanamycin resistance gene for selection. The genes used encode for oxalate oxidase from Triticum aestivum, and for oxalate decarboxylase from Trametes versicolor.

The effectiveness of both constructs was demonstrated in tomato or tobacco plants, where in transformed plants a reduced spreading of the fungus was recorded (Walz et al., 2008 a and b). Similarly, the effectiveness of oxalate oxidase transformation in enhancing resistance to stem rot has been demonstrated in for example soybean by Donaldson et al. (2001)while the effectiveness of oxalate decarboxylase has been demonstrated by Dias et al. (2006) in lettuce.

For detection of both genes, PCR assays with specific primers related to these genes have been developed. The transformation was performed using Agrobacterium tumefaciens under the guidance of Dr. Christian Möllers (Georg-August-Universität Goettingen Department of Crop Sciences, Division of Pant Breeding). After transformation, genetically modified plants were regenerated. Until now some of the plants are still growing on MS medium, while most of them have been transplanted into soil. These plants will be used as resistant standards in the greenhouse and laboratory in order to verify the screening assays and as test genotypes to get a better understanding about the role of oxalic acid in the infection process.

Investigators

Tobias Wulf (Doktorand), Kerstin Höch (Doktorandin)

Supervisor

Prof. Andreas von Tiedemann

Supporting institution

References

Pope, S. J.; Varney, P. L., Sweet, J. B. (1989) Susceptibility of cultivars of oilseed rape to S. sclerotiorum and the effect of infection on yield. Production and protection of oilseed rape and other brassica crops. Aspects of Applied Biology 23, 451-456
Dias, B. B. A., Cunha, W. G., Morais, L. S., Vianna, G. R., Rech, E. L., de Capdeville, G. and Aragão, F. J. L. (2006) Expression of an oxalate decarboxylase gene from Flammulina sp. in transgenic lettuce (Lactuca sativa) plants and resistance to Sclerotinia sclerotiorum. Plant Pathology 55, 187-193.
Donaldson, P. A., Anderson, T., Lane, B. G., Davidson A. L. and Simmonds, D. H. (2001) Soybean plants expressing an active oligomeric oxalate oxidase from the wheat gf-2.8 (germin) gene are resistant to the oxalate-secreting pathogen Sclerotina sclerotiorum. Physiological and Molecular Plant Pathology 59(6), 297-307.
Dunker, S. & Tiedemann, A. v. (2004) Disease/yield loss analysis for Sclerotinia stem rot in winter oilseed rape. IOBC wprs Bulletin (2004) 'Integrated Control in Oilseed Crops', vol. 27 (10), 59-66.
Walz, A., Zingen-Sell, I., Loeffler, M. & Sauer, M. (2008a)Expression of an oxalate oxidase gene in tomato and severity of disease caused by Botrytis cinerea and Sclerotinia sclerotiorum. Plant Pathology 57(3): 453-458
Walz, A., Zingen-Sell, I., Theisen, S. & Kortekamp, A. (2008b) Reactive oxygen intermediates and oxalic acid in the pathogenesis of the necrotrophic fungus Sclerotinia sclerotiorum. European Journal of Plant Pathology 120 (4): 317-330