Derivation of Mutants of Erwinia carotovora subsp. betavasculorum Deficient in Export of Pectolytic Enzymes with Potential for Biological Control of Potato Soft Rot

Abstract

Erwinia carotovora subsp. betavasculorum Ecb168 produces an antibiotic(s) that suppresses growth of the related bacterium Erwinia carotovora subsp. carotovora in culture and in wounds of potato tubers. Strain Ecb168 also produces and secretes pectolytic enzymes and causes a vascular necrosis and root rot of sugar beet. Genes ( out ) involved in secretion of pectolytic enzymes by Ecb168 were localized to two Hin dIII fragments (8.5 and 10.5 kb) of Ecb168 genomic DNA by hybridization to the cloned out region of E. carotovora subsp. carotovora and by complementation of Out - mutants of E. carotovora subsp. carotovora. Out - mutants of Ecb168, which did not secrete pectate lyase into the culture medium, were obtained when deletions internal to either Hin dIII fragment were introduced into the genome of Ecb168 through marker exchange mutagenesis. Out - mutants of Ecb168 were complemented to the Out + phenotype by introduction of the corresponding cloned Hin dIII fragment. Out - mutants of Ecb168 were less virulent than the Out + parental strain on potato tubers. Strain Ecb168 and Out - derivatives inhibited the growth of E. carotovora subsp. carotovora in culture, indicating that the uncharacterized antibiotic(s) responsible for antagonism was exported through an out -independent mechanism. Strain Ecb168 and Out - derivatives reduced the establishment of large populations of E. carotovora subsp. carotovora in wounds of potato tubers and suppressed tuber soft rot caused by E. carotovora subsp. carotovora.