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.IMPORTANCERegulated plant pathogenic bacteria that are subjected to quarantine restrictions play a vital role in the global trade of agricultural products. Plant protection authorities and quarantine inspectors need sensitive, dependable, cost-effective, and easy-to-use, straightforward methods to monitor the status of regulated bacterial agents either in the field or in transportation ports. Clavibacter species are the most destructive gram-positive bacterial plant pathogens. However, isolation and identification of these pathogens are affected by their slow-growing habit on general bacteriological culture media. Until 2015, only five Clavibacter lineages had been identified infecting alfalfa, maize, potato, tomato, and wheat. Within the past decade, however, the range of plant-associated Clavibacter members has been expanded to 13 standalone species. Selective or semi-selective culture media that have so far been developed for the isolation of Clavibacter targeted only the five original pathogens. Thus, application of those culture media nowadays would leave the expanded range of Clavibacter species untargeted. In this study, we aimed to develop a semi-selective medium to support the growth of all Clavibacter lineages while combating the growth of gram-negative bacteria, non-target gram-positive bacteria, and fungi. Furthermore, we designed a PCR primer strictly specific for Clavibacter members with no amplification in the other bacteria or any other living organism. Supplementing the results of semi-selective medium and specific PCR primers led to setting up a reliable, easy-to-use laboratory method for the isolation and identification of Clavibacter species.