Synergetic effect of non-complementary 5’ AT-rich sequences on the development of a multiplex TaqMan real-time PCR for specific and robust detection of Clavibacter michiganensis and C. michiganensis subsp. nebraskensis

Adriana Larrea-Sarmiento; Anne M. Alvarez; James P. Stack; Mohammad Arif

The amplicon generated using either P16S or CM-dnaA primer sets exhibited 100% query coverage and 100% identity against the sequence corresponding to species/subspecies sequence present in NCBI the nucleotide and genome databases, suggesting that the novel primer sets are able to accurately identify plant-associated bacteria as well as the nine subspecies within C. michiganensis. (Fig 3) . Additionally, the axis on the color-coded matrix confirmed the diversity within C. michiganensis subspecies ranging from 89 to 98% (Fig 3) . Fig 3. dnaA color-coded demarcation matrix of Clavibacter michiganensis subspecies. maximum likelihood (ML) phylogeny of 43 partial sequences within C. michiganensis subpecies were used for constructing the pairwise similarity scores, displaying a color-coded matrix. dnaA phylogeny within C. michiganensis shows evolutionary distances gathering nine evident clades, each one representing one subspecies. Bootstrap values were computed with 1,000 replicates using MEGA 7.0.25 and dnaA sequences of Leifsonia xyli and Rathayibacter triciti served as outgroup, sharing less than 89% similarity.