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Leptosphaeria maculans (Phoma lingam) Stem Canker


 
Resistance to stem canker is an important character of winter oilseed rape cultivars. The disease can cause yield losses of up to 40%, and recent average losses in the UK are estimated to be around £30 million each year. The causal fungus, L maculans, exists as two distinct pathotypes, virulent and avirulent. The former leads to the most damaging crown and stem cankers, while the latter induces less damaging pith necrosis.

The disease cycle begins with the formation of leaf spots in the autumn. From these, the virulent pathotype grows systemically down leaf petioles to the stem base, where cankers are formed the following summer. Resistance is currently assessed in trials by spreading infected oilseed rape stem debris in plots, and then assessing canker symptoms on the outside and inside of the stem during June. The procedure is labour intensive, and thus costly, and though the technique is well established and used by plant breeders there are several technical disadvantages. Firstly, the symptoms of stem canker can be difficult to distinguish from those caused by stem infection of light leaf spot, and from insect damage caused by cabbage stem flea beetle. Secondly, the degree of internal blackening recorded often depends on where the stem is cut. An assessment system which evaluated petiole colonisation might offer a more efficient and accurate prediction of cultivar resistance.

PCR probes for the detection and quantification of highly and weakly virulent Leptosphaeria maculans in planta.
 
Published sequence information was used to develop a new set of primers specific for the highly (HV) and weakly (WV) virulent types. Primers HVF1 & HV26c are specific for the highly virulent type whilst WVF1 and WVR1 237 detect the weakly virulent type producing a 377 or 237 base pair product respectively. DNA from 26 isolates of L. maculans collected from UK cultivar trials was extracted and tested with each primer set. None of the isolates tested gave amplification with both primer sets confirming the specificity of the newly designed probes. The majority of isolates were found to be the highly virulent type.  The primers can be used in a multiplex reaction, and the relative proportion of each type using LightCycler melting curve peak comparisons (Fig 1).

              
 

 
Relationship between petiole colonisation and field canker assessments.
L maculans DNA was extracted from petioles of 10 varieties of oilseed rape collected from inoculated field trials in the autumn. The varieties were selected to have a range of canker susceptibility based on extensive data from field records. Petioles were separated into four segments before extraction. DNA levels from segments furthest from the leaf lamina gave the best correlation (r = 0.72) with field scores.

 Predicting canker index from petiole colonisation.

Using data from the 10 varieties, a regression equation was calculated to predict canker index from DNA levels in the petioles of a further 30 varieties.

Predicted index was significantly correlated with field scores (three trial mean), r = 0.61, Fig 2.


              
 

Conclusions
The redesigned primers detected both virulence types, with all isolates screened being designated either highly virulent or weakly virulent type. The amount of pathogen DNA of the highly virulent, canker causing type, in the lower half of the petiole was correlated with observed field resistance to Leptosphaeria maculans. DNA levels in the petiole predicted field canker indices in new varieties with a reasonably high degree of accuracy, though further work is necessary to optimise petiole sampling times in the autumn.

The financial support of DEFRA is gratefully acknowledged.