A pathogenic R. solanacearum strain VL-1 characterized by 16S
rRNA nucleotide sequence (GenBank Accession No. FJ007371) was
grown on Triphenyl Tetrazolium Chloride (TTC) medium for 48 h at
30 C (Kelman, 1954; Kado and Heskett, 1970; Nesmith and Jenkins,
1979). Cultures were maintained in the dark in an incubator, and
were subcultured once a week. Infiltrations of the intracellular
spaces of tobacco plant leaves (15 days old) with suspensions of
bacteria (108 colony forming units (cfu) ml1) were performed
monthly to prove the pathogenicity of pure cultures based on the
development of necrosis (Klement et al., 1964).
The virulence of this strain in tomato was assessed in greenhouse
tests by stem-inoculating tomato plants (cv. Money-Maker)
at the 4-leaf stage with 30 ml of a bacterial suspension containing
108 cfu ml1 (Zanón and Jordá, 2008). After inoculation, plants were
maintained under greenhouse conditions at 18e30 C. Symptoms
of bacterial wilt were assessed 15 days after inoculation. The
presence of the bacterium was confirmed using a Double Antibody
Sandwich-Enzyme Linked Immunosorbent Assay (DAS-ELISA) with
the Loewe commercial antiserum No. 07056 (Loewe Biochemica
GmbH, Sauerlach, Germany).