3.3.34
APPLICATION OF MOLECULAR METHODS TO THE FORECASTING OF SEVERE STEM CANKER EPIDEMICS IN WINTER OILSEED RAPE IN THE UK

RH WILLIAMS and BDL FITT

IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, UK

Background and objectives
Leptosphaeria maculans Desm. [Ces. Et de Not.] (anamorph: Phoma lingam [Tode ex Fr.] Desm.), an ascomycete of worldwide distribution, is the causal agent of stem canker or blackleg disease in a range of crucifers including oilseed rape. In the UK, if meteorological conditions are favourable for ascospore release and disease establishment on plants during the autumn, stem canker can cause substantial yield losses in winter oilseed rape. Early detection of leaf infection is important for effective chemical control. Detection of air-borne ascospores may also be helpful in developing a forecasting strategy to optimise fungicide inputs. Currently, Burkard spore samplers are used to monitor concentrations of air-borne ascospores, thereby necessitating the time consuming visual examination of air spora collected on strips of sticky tape. Moreover, although all isolates of L. maculans appear identical in spore morphology, they can be assigned to one of two groups on the basis of characteristics such as virulence, sirodesmin production and isozyme analysis [11. In general, weakly virulent (WV) isolates do not produce sirodesmins and exhibit isozyme profiles that differ from those of highly virulent (HV), sirodesmin-producing, isolates. However, whilst it appears that only HV isolates cause damaging stem cankers, it is difficult to distinguish cotyledon and leaf lesions caused by the different isolate types. Consequently, it is desirable to develop a rapid method for detection and differentiation of WV and HV isolates of L. maculans in planta or as air-borne spores. Primers for use in the polymerase chain reaction (PCR) have been designed to amplify DNA from either WV or HV isolates [2]. However, their usefulness for differentiating between WV and HV isolates in UK winter oilseed rape crops has not been assessed. It is also unclear whether the PCR could be used in a system for detecting air-borne spores. Therefore, the objectives of this study are to determine whether the PCR, using existing isolate-type specific primers, could be usefully employed to detect HV L. maculans as part of a forecasting scheme to optimise fungicide control of stem canker in winter oilseed rape in the UK.

Results and conclusions
Existing PCR protocols, using isolate-type specific primers, are being assessed for their ability to distinguish between WV and HV UK isolates of L. maculans in planta. The use of these methods to obtain information about the distribution of the pathogen in crops will be investigated. The use of a PCR-based assay to differentiate between ascospores of WV and HV isolates in vitro is being examined. If this proves possible, methods for collecting spores, which are compatible with subsequent DNA amplification, will be developed.

References
1. Williams PH, 1992. Canadian Journal of Plant Pathology 14, 30-35.
2. Mahuku GS, Hall R, Goodwin PH, 1996. European Journal of Plant Pathology 102, 569-576.