Xanthomonas campestris pv. campestris Requires a Functional pigB for Epiphytic Survival and Host Infection

Abstract

When cauliflower plants (Brassica oleraceae) were misted with bacterial suspensions of Xanthomonas campestris pv. campestris (causal agent of black rot of cruciferous plants), two separate populations of the pathogen were associated with the leaves. Initially, bacteria removable by sonication and sensitive to sodium hypochlorite treatment predominated (easily removable epiphytic bacteria, EREB). However, after 2 weeks, bacteria not removable by sonication and insensitive to sodium hypochlorite treatment were dominant. Although the exact location of this second population of the pathogen was not determined, evidence is presented to support its location in protected sites on the leaf surface. pigB of this pathogen is required for production of extracellular polysaccharide (EPS), xanthomonadin pigments, and the diffusible signal molecule, DF (diffusible factor). DF can extracellularly restore EPS and xanthomonadin production to pigB mutant strains. Parent strain B-24 and pigB mutant strain B24-B2 were identical for in planta growth and symptomatology after artificial infection by injection in leaf mid-veins. Subsequently, X. campestris pv. campestris parent strain B-24, Tn3HoHo1 pigB insertion mutation strain B24-B2, chromosomally restored pigB mutation strain B24-B2R, and strain B24-79 with a Tn3HoHo1 insertion in an unrelated part of the genome were compared for epiphytic survival on, and natural infection of, cauliflower. After application, strains B-24, B24-B2R, and B24-79 all maintained leaf EREB populations of between approximately 3 and 6 (log [1 + CFU per g of fresh weight]) over a 3-week period, whereas B24-B2 populations fell to nearly undetectable levels. Plants sprayed with strains B-24, B24-B2R, and B24-79 averaged between 1.0 and 1.2 lesions, whereas those sprayed with B24-B2 averaged only 0.03 lesions per plant after 3 weeks. Differences in EREB population levels did not explain the observed differences in host infection frequencies, and the results indicated that strain B24-B2 was reduced in its ability to infect the host via the hydathodes, but unaffected in infection via wounds. When strains B-24 and B24-B2 were mixed in equal numbers and sprayed on plants together, B24-B2 epiphytic populations were intermediate between those of B-24 applied alone and B24-B2 applied alone. These results indicate that a functional pigB is required for epiphytic survival and natural host infection under the experimental conditions tested, and suggest that DF, xanthomonadins, and EPS could all be important for survival of this pathogen on the leaf surface, and/or for host infection.