1.1.23
THE LEPTOSPHAERIA MACULANS:BRASSICA NAPUS INTERACTION IN AUSTRALIA

TJ WIECHEL1, GJ ASH1, N WRATTEN2 and CE MAY2

1School of Agriculture, Charles Sturt University, Wagga Wagga, New South Wales, Australia 2678; 2New South Wales Agriculture, Agricultural Research Institute, Wagga Wagga, New South Wales, Australia 2650

Background and objectives
Blackleg of canola (Brassica napus), caused by the fungus Leptosphaeria maculans, is a serious disease in Australia. The initial infection is via a leaf lesion, the fungus then spreads down the petiole (biotrophic phase) and invades the stem cortex (necrotrophic phase), resulting in a basal stem canker which causes the plant to lodge and die [1]. The current methods of control are resistant varieties and cultural practices. The mechanisms of resistance are not understood, and current breeding for resistance is centred on stem canker resistance. The consistent development of resistant cultivars requires an understanding of the pathogenic variation of the fungus and its ability to overcome the resistance. The pathotype structure of a plant pathogen can be determined using a set of differentials with known gene differences. Isolates of L. maculans in the northern hemisphere are currently grouped into four pathotypes (PG1-4) based on their ability to cause lesions on the cotyledons of commerical cultivars Westar, Glacier and Quinta [2]. There are several problems associated with this differential set: the pedigree of the cultivars is known but the specific gene differences have not been determined; these cultivars are not homozygous so during selfing and seed increase the differential gene may not be present in the resulting seed used for screening. The use of doubled haploid (DH) lines, theoretically overcomes the problems associated with heterozygosity; seed can be maintained and each seed should be identical and contain the differential gene of interest. The aim of this study is to establish a set of homozygous differentials for blackleg resistance in canola; based on these differentials, to characterize race (pathotype) variation in L. maculans; and to determine the likely number and heritability of markers for virulence using RAPD and AFLP analysis.

Results and conclusions
In 1995, 486 DH canola lines were assessed for blackleg resistance under varying inoculum pressure in a blackleg nursery and yield paddock in Wagga Wagga. The disease symptoms were classified as healthy, moderate canker and severe canker. One hundred and twenty of these lines with varying resistance were selected for glasshouse pathogenicity trials at the cotyledon stage, using six isolates ranging in virulence. All isolate x line combinations produced lesions, no lines were immune. Only 12 lines differentiated N10, a weakly virulent isolate, from the other isolates which produced small grey sunken lesions with thick black edges. The remaining isolates produced lesions with brown centres and grey-green diffuse edges. Forty lines did not differentiate the isolates, whereas the remaining lines differentiated to a small extent statistically but not visually. Twenty six of those DH lines are currently being assessed for differential reactions at the adult stage to determine if backleg resistance at the cotyledon and adult stage are under different genetic control. The six isolates have been analysed with 150 RAPD primers: 83 primers have produced RAPD profiles but only 33 primers have produced profiles which detect differences that may be linked to virulence. AFLP screening has detected 10 primer combinations which produce polymorphisms. N10 (weakly virulent) is differentiated by three primer combinations while N13 (moderately virulent) is differentiated by seven primer combinations. AFLP analysis has been more effective at differentiating the isolates. Matings have been performed between a highly virulent isolate (WA) and a weakly virulent isolate (N10), and 17 progeny are currently being assessed for the inheritance of specific virulence markers. The inheritance of these markers will provide information on the likelihood of new races arising in the field.

References
1. Hammond KE, Lewis BG, 1986. Physiological and Molecular Plant Pathology 28, 251-65.
2. Williams PH, 1992. Canadian Journal of Plant Pathology 14, 30-35.