Isolation and identification of Clavibacter made easy

Clavibacter species are the most important gram-positive bacterial plant pathogens in terms of both disease severity and economic importance. As a group of regulated and quarantined plant pathogens, Clavibacter members are slow-growing organisms on general culture media, leading to difficulties in their isolation and identification. In this study, we developed an efficient, straightforward method for the isolation and identification of Clavibacter members from plant materials. First, a semi-selective medium was developed from a modification of conventional YPGA and YDC media, namely ClaYPGA and ClaYDC, respectively. This semi-selective medium contained four antibiotics, that is , colistin (100.0 mg/L), nalidixic acid (5.0 mg/L), sulfamethoxazole (5.0 mg/L), and trimethoprim (5.0 mg/L) for the inhibition of non-target bacterial growth, as well as natamycin (500.0 mg/L) for the inhibition of fungal growth. The ClaYPGA/ClaYDC medium supported the growth of all Clavibacter species while inhibiting the growth of almost all gram-negative bacteria, gram-positive Bacillus and Streptomyces , and fungi. Furthermore, ClaYPGA and ClaYDC did not affect the biological features, that is , cell density, and colony morphology of Clavibacter species compared to the basal YPGA and YDC. To increase the specificity of semi-selective medium, the primer pair ClaFor/ClaRev was designed based on the nucleotide sequence of a 259 bp DNA fragment found only in Clavibacter members. These PCR primers were strictly specific to Clavibacter species. The combination of semi-selective medium and ClaFor/ClaRev PCR primers increased the precision of the isolation and identification of Clavibacter species to such an extent that it has never been reported in the literature.