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 gram-positive aerobic species Clavibacter michiganensis, which resides in the xylem vessels of the host, is a devastating bacterial pathogen of many economically important agricultural crops. Currently, the genus Clavibacter is known only for one species, C. michiganensis which belongs to family Microbacteriaceae within the phytopathogenic coryneform group, known for epi-or endophytic colonization of symptomatic as well as several asymptomatic plant species [1, 2] . Virulence genes within this species are plasmid-borne (not essentially present), while host colonizing genes are chromosomal [3] . Hitherto, nine subspecies based on host specificity and bacteriological characteristics have been identified within the single species of C. michiganensis. C. michiganensis subsp. nebraskensis is the causal agent of Goss's bacterial wilt and blight of corn (Zea mays), which is listed among the five most significant diseases of corn in the U.S. Goss's wilt accounts for high yield losses in the Great Plains and Midwest regions of the corn belt of the U.S., the largest producer and exporter of corn worldwide. Since the first report in 1969 from Nebraska, the disease has been spreading and adversely impacting corn production in the Midwestern United States including western Indiana, Illinois, Iowa, Missouri, eastern Nebraska, and eastern Kansas, as well as Canada. Goss's disease shows two major types/phases of symptoms -a leaf blight and a systemic vascular wilt [6, 7, 8] . The leaf blight phase involves leaves and above-ground parts of the plant which exhibit small, dark and discontinuous watersoaked spots; orange shiny bacterial exudates are also observed in advanced stages. The less common wilt phase involves systemic infection of xylem elements resulting in discoloration of vascular bundles, stalk rot and premature plant death. Transmission of Goss's disease usually occurs through open wounds on leaves, and C. m. subsp. nebraskensis can overwinter and remain in plant debris to serve an inoculum for the next crop; transmission through seeds is minimal [2, 8, 9] .