XANTHOMONAS-CAMPESTRIS PV. CAMPESTRIS REQUIRES A FUNCTIONAL PIGB FOR EPIPHYTIC SURVIVAL AND HOST INFECTION

When cauliflower plants (Brassica oleraceae) were misted with bacteria l suspensions of Xanthomonas campestris pv. campestris (causal agent o f black rot of cruciferous plants), two separate populations of the pa thogen were associated with the leaves. Initially, bacteria removable by sonication and sensitive to sodium hypochlorite treatment predomina ted (easily removable epiphytic bacteria, EREB). However, after 2 week s, bacteria not removable by sonication and insensitive to sodium hypo chlorite treatment were dominant. Although the exact location of this second population of the pathogen was not determined, evidence is pres ented to support its location in protected sites on the leaf surface, pigB of this pathogen is required for production of extracellular poly saccharide (EPS), xanthomonadin pigments, and the diffusible signal mo lecule, DF (diffusible factor). DF can extracellularly restore EPS and xanthomonadin production to pigB mutant strains. Parent strain B-24 a nd pigB mutant strain B24-B2 were identical for in planta growth and s ymptomatology after artificial infection by injection in leaf mid-vein s. Subsequently, X. campestris pv. campestris parent strain B-24, Tn3H oHo1 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 surviva l on, and natural infection of, cauliflower. After application, strain s B-24, B24-B2R, and B24-79 all maintained leaf EREB populations of be tween approximately 3 and 6 (log [1 + CFU per g of fresh weight]) over a 3-week period, whereas B24-B2 populations fell to nearly undetectab le levels. Plants sprayed with strains B-24, B24-B2R, and B24-79 avera ged between 1.0 and 1.2 lesions, whereas those sprayed with B24-B2 ave raged only 0.03 lesions per plant after 3 weeks. Differences in EREB p opulation levels did not explain the observed differences in host infe ction frequencies, and the results indicated that strain B24-B2 was re duced in its ability to infect the host via the hydathodes, but unaffe cted in infection via wounds. When strains B-24 and B24-B2 were mixed in equal numbers and sprayed on plants together, B24-B2 epiphytic popu lations were intermediate between those of B-24 applied alone and B24- B2 applied alone. These results indicate that a functional pigB is req uired for epiphytic survival and natural host infection under the expe rimental conditions tested, and suggest that DF, xanthomonadins, and E PS could all be important for survival of this pathogen on the leaf su rface, and/or for host infection.