Introduction Phoma stem canker is an economically important disease that causes considerable yield loss in oil- seed rape (Brassica napus L.) every year world- wide (Fitt et al. 2006). It is associated with a complex of fungal pathogens, comprising 2 spe- cies, Leptosphaeria maculans (Desm.) Ces. et de Not. and L. biglobosa (Shoemaker and Brun 2001). All parts of the susceptible oilseed rape host can be colonized by these pathogens, includ- ing the tap root, stem base, upper stem parts, leaves, cotyledons, and seeds (Paul and Rawlinson 1992). In Europe, the first disease symptoms on winter oilseed rape appear in autumn, when coty- ledons and leaves are inoculated naturally with airborne ascospores (sexual spores), which germi- nate to penetrate and cause phoma leaf spot lesions (West et al. 1999; Toscano-Underwood et al. 2003). Thereafter, mycelia of the pathogens colo- nise host tissues biotrophically, through endophytic growth within the petiole, to reach the stem where stem lesions and damaging basal stem cankers are produced (Hammond et al. 1985). In Europe, crop disease control consists mostly in the use of resistant cultivars, stubble manage- ment, and fungicide application (Gladders et al. 1998; Li et al. 2003; Rouxel et al. 2003). As asco- spores and splash-dispersed pycnidiospores (asex- ual spores) constitute inocula in phoma disease epidemics, information on timing of airborne propagule release is an essential tool in the protec- tion of oilseed rape against Leptosphaeria spp. (Gladders et al. 1998; West et al. 2002). The effec- tiveness of chemical control of this disease is de- pendent on both the timing and dosage of fungicide application. In Poland, ascospore re- lease is monitored by the System for Forecasting J Appl Genet 50(4), 2009, pp. 411–419 Original article Analyses of air samples for ascospores of Leptosphaeria maculans and L.biglobosa by light microscopy and molecular techniques J. Kaczmarek 1 , M. Jêdryczka 1 , B.D.L. Fitt 2 , J.A. Lucas 2 , A.O. Latunde-Dada 2 1 Institute of Plant Genetics, Polish Academy of Sciences, Poznan, Poland 2 Rothamsted Research, Harpenden, United Kingdom Abstract. Spores of many fungal pathogens are dispersed by wind. Detection of these airborne inocula is impor- tant in forecasting both the onset and the risk of epiphytotics. Species-specific primers targeted at the internal transcribed spacer (ITS) region of Leptosphaeria maculans and L. biglobosa – the causal organisms of phoma stem canker and stem lesions of Brassica spp., including oilseed rape – were used to detect DNA extracted from particles deposited on tapes obtained from a spore trap operated in Rarwino (northwest Poland) from September to November in 2004 and 2006. The quantities of DNA assessed by traditional end-point PCR and quantitative real-time PCR were compared to microscopic counts of airborne ascospores. Results of this study showed that fluctuations in timing of ascospore release corresponded to the dynamics of combined concentrations of DNA from L. maculans and L. biglobosa, with significant positive correlations between ascospore number and DNA yield. Thus the utilization of PCR-based molecular diagnostic techniques enabled the detection, identification, and accurate quantification of airborne inoculum at the species level. Moreover, real-time PCR was more sensi- tive than traditional PCR, especially in years with low ascospore numbers. Keywords: ascospore release, disease forecasting, molecular biological diagnostics, oilseed rape, phoma stem canker, polymerase chain reaction, real-time PCR, spore trapping. Received: February 26, 2009. Accepted: May 22, 2009. Correspondence: J. Kaczmarek, Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyñska 34, 60–479 Poznañ, Po- land; e-mail: jkac@igr.poznan.pl