Comparative Genomics to Develop a Specific Multiplex PCR Assay for Detection of Clavibacter michiganensis

Shree P. Thapa, Michael O’Leary, Marie-Agnès Jacques, Robert L. Gilbertson, and Gitta Coaker

BACTERIOLOGY J. - Published Online:31 Jan 2020 https://doi.org/10.1094/PHYTO-10-19-0405-R

Abstract

Clavibacter michiganensis is a Gram-positive bacterial pathogen that proliferates in the xylem vessels of tomato, causing bacterial wilt and canker symptoms. Accurate detection is a crucial step in confirming outbreaks of bacterial canker and developing management strategies. A major problem with existing detection methods are false-positive and -negative results. Here, we report the use of comparative genomics of 37 diverse Clavibacter strains, including 21 strains sequenced in this study, to identify specific sequences that are C. michiganensis detection targets. Genome-wide phylogenic analyses revealed additional diversity within the genus Clavibacter. Pathogenic C. michiganensis strains varied in plasmid composition, highlighting the need for detection methods based on chromosomal targets. We utilized sequences of C. michiganensis-specific loci to develop a multiplex PCR-based diagnostic platform using two C. michiganensis chromosomal genes (rhuM and tomA) and an internal control amplifying both bacterial and plant DNA (16s ribosomal RNA). The multiplex PCR assay specifically detected C. michiganensis strains from a panel of 110 additional bacteria, including other Clavibacter spp. and bacterial pathogens of tomato. The assay was adapted to detect the presence of C. michiganensis in seed and tomato plant materials with high sensitivity and specificity. In conclusion, the described method represents a robust, specific tool for detection of C. michiganensis in tomato seed and infected plants.

See https://apsjournals.apsnet.org/doi/10.1094/PHYTO-10-19-0405-R

Figure 3: Clavibacter michiganensis primer design and multiplex PCR. A, Genetic architecture of flanking genes surrounding the tomA and rhuM loci. The chp/tomA cluster, including tomA, is only present in pathogenic C. michiganensis. rhuM is only present in C. michiganensis tomato pathogens. Primers used to amplify each gene are shown in gray. B, Agarose gel electrophoresis of multiplex PCR assay products. Total genomic DNA was used as a template with the RhuM-F/R primers targeting the rhuM gene, the TomA-F1/R1 primers targeting the tomA gene, and the 16sR-F/R primers targeting 16S ribosomal RNA (rRNA) in either bacteria or plants. Lanes 1 to 5, endophytic Clavibacter strains (CASJ009, CFBP8017, CFBP8019, CFBP7576, and CFBP7494); lanes 6 to 39, pathogenic C. michiganensis (CFBP7311, CFBP1940, CFBP5842, CFBP7568, CFBP7488, CFBP2500, CFBP7158, CFBP7312, CFBP7316, CFBP7314, CFBP6885, CFBP7315, CFBP7589, NCPPB382, ATCC14456, ATCC10206, NZ2541, NZ1811, NZ5026, CASJ001, CASJ002, CASJ003, CASJ004, CASJ005, CASJ008, CA00001, CAYO001, AZ-28, NZ2550, 285D, 12B, 13C1, Cm4, and Cm5); lanes 40 to 43, C. sepedonicus (NZ2535, SB109, INM-1, and BRR7); lane 44, C. insidiosus (CIC266); lanes 45 and 46, C. tessellarius (CIC021 and CIC022); lanes 47 and 48, Xanthomonas euvesicatoria (RL677 and HB1); lanes 49 to 51 Pseudomonas syringae pv. tomato (PstA9, Pst18, and Pst838-8); lanes 52 to 54, Escherichia coli, ‘Candidatus Liberibacter asiaticus’ HHCA, and uninfected tomato DNA; and lane M = 1-kb DNA ladder.