Dissecting Botrytis cinerea pathogenicity using a non-pathogenic mutant

Caroline Kunz (1), Benoit Poinssot (2), Alain Pugin (2), Martine Boccara (1)
(2) Laboratoire de Pathologie Végétale INA/PG, UMR 217 INRA-INA/PG-Paris VI, 16 rue Claude Bernard, 75231 Paris cedex 05
(3) UMR INRA/Université de Bourgogne, Laboratoire de Biochimie, Biologie cellulaire et Ecologie des Interactions Plantes/Micro-Organismes, INRA, 17 rue Sully, BP 86510, 21065 Dijon cedex, France.

The ascomycete Botryotinia fuckeliana (anamorph Botrytis cinerea) is an economically important, necrotrophic plant pathogen, which attacks a broad number of plants. To obtain more insight into B. cinerea pathogenicity we used insertionnal mutagenesis to generate a collection of mutants with reduced or no pathogenicity. We have focused our studies on the non-pathogenic mutant A336, which presents a monogenetic phenotype. Nevertheless, the mutation is not tagged by the pAN7 plasmid used for mutagenesis.
Mutant A336 grows normally on acidic pH (pH 5) but grows poorly at pH 7. We have shown that the mutant lacks production of oxalic acid and is incapable of acidifying culture medium originally at elevated pH.
When inoculated onto grapevine leaves, mutant A336 induces small necrotic lesions, not observed for wild type strain Bd90. When observed under fluorescent microscopy, these necrotic lesions reflect the production of fluorescent (phenolic) compounds. Culture supernatants of A336 and Bd90 were tested for elicitation on vine leaf cell cultures. When grown on Czapek glucose medium at pH 6, A336 supernatant showed high elicitation activity, whereas no such activity was detected for wild type Bd90. The oxidative burst induced by A336 supernatant was repressed in the presence of 10 mM oxalic acid. When grown on Nitsch-Nitsch medium at pH 5.6, supernatants of both strains showed elicitation activity. We are currently analysing the two culture media in order to identify the origin of the induction or repression of an elicitor production in wild type strain Bd90. An elicitor has recently been purified from B. cinerea by Poinssot et al. (submitted).
To try to complement the non-pathogenic phenotype of A336 the mutant and wild type strain Bd90 were grown on culture agar medium supplemented with 10 mM oxalic acid, which is not produced by the mutant (see above), before applying mycelium plugs to grapevine leaves. No phenotype complementation was achieved in this way and mutant A336 stayed non-pathogenic on wounded or intact leaves and, moreover, did no longer provoke micro-lesions. Interestingly, wild type strain Bd90 lost its pathogenicity on intact grapevine leaves, when grown on oxalic acid containing medium.
The role of oxalic acid and elicitor activity in B.cinerea pathogenicity will be discussed and a model taking into consideration our results and general knowledge on B. cinerea pathogenicity will be presented.