Colonization of siliques and seeds of rapid cycling Brassica oleracea plants by Xanthomonas campestris pv. campestris after spray-inoculation of flower clusters

Glasshouse experiments were conducted to study the colonization of seedpods (siliques) and seeds of rapid cycling Brassica oleracea plants after spraying inoculum on clusters of recently opened flowers with Xanthomonas campestris pv. campestris (Xcc) at densities of 107–108 cfu ml−1. A green fluorescent protein (GFP) tagged Xcc strain was used to allow visualization of the bacteria by epifluorescence stereo microscopy (ESM) and confocal laser scanning microscopy (CLSM). The GFP-tagged strain showed reduced virulence compared to the untagged parental strain, but was still able to cause black rot symptoms. Two to three days after spray-inoculation, sepals, stamen and petals were colonized by Xcc, as observed by ESM. In green siliques a GFP-signal was observed on valves, septa and seeds, despite the fact that a high percentage of Xcc cells had lost their ability to express GFP as found by dilution-plating. Densities of Xcc in infected silique tissues were up to 109 cfu g−1. A fluorescent signal using ESM was found in seeds harvested from symptomatic siliques after incubation of seeds on blotting paper wetted with broth to enhance the multiplication of Xcc. Xcc was found in association with the seed coat and in a single seed, also in the endosperm and embryo, indicating deep-seated seed infection. The estimated incidence of contaminated seeds in both years was ca. 7%. The estimated incidence of deep-seated infections, still detectable after warm water treatment of seeds, was also high (2–3.8%). It is concluded that spray-inoculation of flower clusters with Xcc can result in the infection of sepals and reproductive organs, and in deep-seated seed infections.