Samples were collected from residential
and commercial citrus as possible
citrus canker lesions from eight counties
(Fig. 1A, Table 1). Diseased tissues were
surfaced-sterilized and crushed in 1 ml of
sterile tap H2O (dH2O) in a test tube using
a glass rod. Samples were spread on nutrient
agar (NA) plates and incubated at 28°C
for 3 days. Whitish colonies of bacteria
producing a diffusible nonfluorescent pigment
were consistently isolated. Isolates
were purified from single colonies and
tested by Gram staining. Isolates were
stored at room temperature in sterile tap
H2O and at –80°C in fresh nutrient broth
containing 15% glycerol. The pathogenicity
of all isolates was first screened by
infiltrating the bacterial suspension in
sterile tap H2O at 108
CFU/ml into leaves
of tomato (Lycopersicon esculentum var.
esculentum), pepper (Capsicum spp.), and
tobacco (Nicotiana tabacum). Isolates producing
an HR on tomato and pepper were
considered pathogenic. A total of 10 isolates
were recovered from infected citrus.
For comparison, B. andropogonis was
isolated from Bougainvillea sp. and from
carnation (Dianthus caryophyllus). The B.
andropogonis type strain (ATCC 23061)
from sorghum (renamed 1976 and kindly
provided by Jeffery Jones, University of
Florida) was also included in many tests
(Table 1).