3.5.17

TRANSPOSON-INDUCED MUTANTS OF PSEUDOMONAS CHLORORAPHIS MA 342 DEFECTIVE IN THE PRODUCTION OF AN ANTIFUNGAL METABOLITE EXERT REDUCED BIOCONTROL ACTIVITY


M HOKEBERG1, SAI WRIGHT1, M SVENSSON 2, L LUNDGREN2 and B GERHARDSONl

1Plant Pathology and Biocontrol Unit, Swedish University of Agricultural Sciences, Uppsala, Sweden; 2Department of Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden

Background and objectives
Application of the biocontrol agent Pseudomonas chlororaphis, strain MA 342, to cereal seeds controls the seed-borne diseases caused by Drechslera teres and Tilletia caries as effectively as conventional fungicides [1]. The strain has exerted biocontrol effect in field experiments also against diseases caused by D. graminea, D. avenae, Ustilago hordei, U. avenae and Microdochium nivale. Culture supernatant of MA 342 contains a secondary metabolise that impedes the growth of D. teres and M. nivale in vitro. To elucidate the importance of this metabolise in the mode of action in biological control, several transposon-induced mutants of MA 342 deficient in the production of this metabolise were tested for biological control efficacy in a greenhouse assay and are currently being characterized genetically. Approximately one thousand randomly generated Tn5 insertion mutants of MA 342 were screened for loss of inhibitory activity to M. nivale by examining the sizes of inhibition zones produced by the mutants against M. nivale on TSA plates.

Results and conclusions
Nine mutants of MA 342 had lost some or all of the activity against M. nivale in vitro. These nine mutants were subjected to a test for biocontrol ability against barley net blotch, caused by D. teres in a greenhouse bioassay, as previously described [1]. Furthermore, the production/lack of production of the antifungal metabolise by the mutants was determined by chemical analysis. The mutants produced none or reduced amounts of the metabolise. The amount of metabolise production was directly correlated to the degree of suppression of D. teres of barley in the greenhouse. Therefore, it seems likely that the antifungal metabolise produced by MA 342 is involved in the biological control activity against barley net blotch in the greenhouse test. The positions of the Tn5 insertions in the nine mutants are being mapped, and the mutants are being classified into distinct groups. At present, we are in the process of constructing a genomic library of MA 342 with the final goal to clone and characterize the genes responsible for the synthesis of the metabolise.

References
1. Hdkeberg M, Gerhardson B, Johnsson L, 1997. European Journal of Plant Pathology 103, 25-33.