Genetic Evidence that Extracellular Polysaccharide Is a Virulence Factor of Pseudomonas solanacearum. Timothy P. Denny. Department of Plant Pathology, The University of Georgia, Athens 30602 U.S.A. Seung-Ryel Baek. Department of Plant Pathology, The University of Georgia, Athens 30602 U.S.A. MPMI 4:198-206. Accepted 27 December 1990. Copyright 1991 The American Phytopathological Society.

To test whether the extracellular polysaccharide (EPS) produced by Pseudomonas solanacearum is responsible for the wilt symptoms caused by this plant pathogen, two classes of Tn5-induced, EPS-impaired mutants were further characterized and manipulated. In comparison with wild-type AW1, the class I mutant AW1-1 produced about 95% less EPS on rich and minimal media and slowly wilted one third as many tomato plants in stem and root inoculation assays. In contrast, class II mutants produced nearly wild-type amounts of EPS on minimal medium and largely retained virulence. Eight cosmid clones were identified in a genomic library of wild-type strain AW1 that restored EPS production by the mutants to varying degrees, with one cosmid fully complementing both class I and class II mutants. Southern blot analysis, restriction mapping, subcloning, and Tn3-HoHo1 mutagenesis demonstrated the existence of two neighboring regions involved in EPS production. The minimum sizes for regions I and II were 9 and 2.6 kilobases (kb), respectively. Phenotype conversion, which results in spontaneous EPS‾ mutants, decreased expression of lacZ fusions in both regions. EPS⁺ merodiploids of AW1-1 were more virulent than AW1-1, but plasmid instability in planta hindered our interpretation of these experiments. However, allelic replacement in certain AW1-1 merodiploids generated strains that were EPS⁺ and kanamycin sensitive, and these wild-type recombinants were invariably as virulent as AW1. These results provide genetic evidence that EPS is an important virulence factor required by P. solanacearum strain AW1 to wilt tomato.