Evaluation of the Role of Syringomycin in Plant Pathogenesis by Using Tn 5 Mutants of Pseudomonas syringae pv. syringae Defective in Syringomycin Production

Abstract

Syringomycin is a necrosis-inducing phytotoxin produced by Pseudomonas syringae pv. syringae. To determine whether syringomycin production is a determinant in virulence or pathogenicity, we isolated nontoxigenic (Tox − ) Tn 5 -containing mutants and then quantitatively evaluated them for the ability to multiply and cause disease in immature sweet-cherry fruits. Transposon Tn 5 was delivered to Tox + strain B301D-R by using the suicide vector, pGS9, and the resultant kanamycin-resistant (Km r ) colonies were screened for changes in syringomycin production by testing for antibiosis against Geotrichum candidum. Southern blot analysis of Kpn I-and Eco RI-digested DNA showed that 15 (0.3%) Tox − mutants were isolated which had Tn 5 inserted into 1 of 14 distinct loci. Phenotypic characterization of the Tox − mutants identified three major groups, which were differentiated by pathogenicity and ability to cause a tobacco hypersensitive reaction (HR). The eight strains in group A were pathogenic (Path + ) in cherry fruit assays, but the disease index was 17 to 66% lower (significant at P = 0.01) than for the parental Tox + strain, B301D-R. The population dynamics of group A strains W4S770 and W4S116 in cherry fruits were, however, indistinguishable from that of strain B301D-R. The remaining seven Tox − strains were nonpathogenic; group B strain W4S2545 (Path − HR + ) and group C strain W4S468 (Path − HR − ) developed significantly lower populations (10 5 to 10 7 CFU per cherry fruit) 3 days after inoculation than strain B301D-R did (nearly 10 9 CFU per fruit). The data indicate that syringomycin is not essential for pathogenicity, but contributes significantly to virulence.