Bacterial head (or spear) rot of broccoli: pathogenicity and cultivar susceptibility
Bacterial head rot of broccoli is an opportunistic infection caused predominantly by Pseudomonas fluorescens. Whilst crop losses of between 30 and 100% have been reported, little is known of the disease aetiology. The research conducted in this thesis covered two main areas: Broccoli cultivar susceptibility to disease and bacterial pathogenicity.Cultivar susceptibility: Two in vitro pathogenicity tests to distinguish disease susceptibility levels were developed. The first test, inoculating seedling hypocotyls with a needle using bacteria removed from an agar colony, produced low disease levels and variability between replicates and therefore was rejected. The second test, based on excised heads from mature plants, successfully distinguished between 10 cultivars with the results corresponding to previously published data for resistance (with one exception). Head morphology, in relation to disease susceptibility, was also studied. Differences in morphology may predispose certain cultivars to disease by influencing the duration free water remains on the head and the availability of sites for infection. Head shape (doming), head size, bud number, bud prominence and stomatal number were assessed. Whilst these factors differed significantly between cultivars, two sets of factors correlated positively at a significant level: Head size and disease susceptibility, and head doming and disease resistance.Pathogenicity: Biosurfactants, produced by pathogenic bacterial strains, have been implicated in the development of head rot on unwounded heads, by aiding bacterial establishment and spread. Their role in disease, as virulence or pathogenicity factors, was assessed using a mutant approach.Initially, five biosurfactant detection tests were evaluated to allow the mutants produced to be tested for loss of production. The most suitable proved to be monitoring changes in the surface tension of a water droplet following the addition of bacterial cells removed from a colonyThirty-five mutants of a Pseudomonas fluorescens strain, defective in biosurfactant production (surf'), were produced by Tn5 mutagenesis. Southern blotting demonstrated that17 of the 35 mutants had single Tn5 insertions, while the rest had more than one insert. Twenty-two different Sail profiles were found in the 35 mutants indicating that 13 mutants shared their Tn5-inserted restriction pattern with at least one other strain. Of the 17 with only one Tn5 insertion, five were reduced in their ability to produce disease symptoms (vir '), whilst the other 12 showed no change. Although vir ' mutants were also detected amongst the18 mutants with multiple insertions, biosurfactant-loss may not be the only cause of reduced virulence, because other gene functions may have been affected. Growth rate and the production of three extracellular products (pectic and proteolytic enzymes, and fluorescent pigment) were unaffected in these 17 mutants. Therefore, because some s u r f ' mutants were unaffected in their ability to cause disease, it was concluded that biosurfactant production in P. fluorescens is neither a virulence nor a pathogenicity factor. Biosurfactants are probably produced, in this bacterium, to assist initial establishment and colonisation of the plant surface. Further work is required to elucidate the nature of the mutated genes to define the precise role of biosurfactants in the host/pathogen interaction.