Genome analysis and development of a multiplex TaqMan real-time PCR for specific identification and detection of clavibacter michiganensis subsp. Nebraskensis

Citation

Tambong, J.T., Xu, R., Daayf, F., Brière, S., Bilodeau, G.J., Tropiano, R., Hartke, A., Reid, L.M., Cott, M., Cote, T., Agarkova, I. (2016). Genome analysis and development of a multiplex TaqMan real-time PCR for specific identification and detection of clavibacter michiganensis subsp. Nebraskensis, 106(12), 1473-1485. http://dx.doi.org/10.1094/PHYTO-05-16-0188-R

Plain language summary

The reemergence of the Goss’s bacterial wilt and blight disease in corn in the United States and Canada has prompted investigative research to better understand the genome organization. The disease is caused by Gram-positive bacteria with yield losses of up to 50% reported. In this study, we we performed genome analysis on new strains isolated in 2014 compared to the strain isolated over 40 years. The results show that the recent strains are different from the 40-year old strain. Based on the genome information, a detection tool was developed for rapid and reliable identification of these bacteria on infected corn leaf tissues. This tool should fill the current gap for a reliable diagnostic technique for these bacteria.

Abstract

The reemergence of the Goss's bacterial wilt and blight disease in corn in the United States and Canada has prompted investigative research to better understand the genome organization. In this study, we generated a draft genome sequence of Clavibacter michiganensis subsp. nebraskensis strain DOAB 395 and performed genome and proteome analysis of C. michiganensis subsp. nebraskensis strains isolated in 2014 (DOAB 397 and DOAB 395) compared with the type strain, NCPPB 2581 (isolated over 40 years ago). The proteomes of strains DOAB 395 and DOAB 397 exhibited a 99.2% homology but had 92.1 and 91.8% homology, respectively, with strain NCPPB 2581. The majority (99.9%) of the protein sequences had a 99.6 to 100% homology between C. michiganensis subsp. nebraskensis strains DOAB 395 and DOAB 397, with only four protein sequences (0.1%) exhibiting a similarity <70%. In contrast, 3.0% of the protein sequences of strain DOAB 395 or DOAB 397 showed low homologies (<70%) with the type strain NCPPB 2581. The genome data were exploited for the development of a multiplex TaqMan real-time polymerase chain reaction (PCR) tool for rapid detection of C. michiganensis subsp. nebraskensis. The specificity of the assay was validated using 122 strains of Clavibacter and non-Clavibacter spp. A blind test and naturally infected leaf samples were used to confirm specificity. The sensitivity (0.1 to 1.0 pg) compared favorably with previously reported realtime PCR assays. This tool should fill the current gap for a reliable diagnostic technique.

Publication date

2016-12-01

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