QUANTIFICATION OF LEPITOSPHAERIA-MACULANS GROWTH IN COTYLEDONS OF BRASSICA-NAPUS USING ELISA

The result of the Leptosphaeria maculans/Brassica napus interaction is usually assessed by symptom scoring following a cotyledon-inoculation test. However, an early evaluation of the interaction, and reliable q uantitative data of fungal growth inside plant tissues are needed to s upplement the visual assessment of the symptoms. For this purpose, we developed a quantitative double antibody sandwich enzyme-linked immuno sorbent assay (DAS-ELISA) using rabbit polyclonal antisera directed ag ainst soluble mycelial proteins. The specificity of the serum was firs t assessed by immunoblotting following isoelectric focusing of soluble proteins (Western blot) and by DAS-ELISA. Except for Alternaria brass icae, no cross-reactions were observed with mycelial extracts of sapro phytes or pathogens of B. napus following DAS-ELISA. Although Tox(+) a nd Tox(0) isolates of L. maculans were unequivocally discriminated by Western blot, they were quantitatively indistinguishable following ELI SA, thus enabling us to analyse a wide range of L. maculans isolates i n planta. The detection limit of the assay was less than 10 ng of fung al proteins per ml of plant extract. For a given isolate, time-course studies showed that fungal growth in cotyledons was correlated with sy mptom scoring. In the case of hypersensitive response, only 34% of the plants were ELISA-positive, and these plants never contained more tha n 10 ng of fungal protein per cotyledon. In contrast, in the cases of susceptibility, 100% of the plants were ELISA-positive and fungal prot ein content was higher than 10 mu g per cotyledon. Moreover, significa nt differences in ability to colonize the tissues were observed among Tox(+) isolates. Finally, using the ELISA quantification, intermediate symptoms could be differentiated as late-resistance responses or susc eptibility.