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

Thus far, no foliar bactericide applications are available for control; therefore, disease resistance/tolerance genotype selection with integrated pest management practices including crop rotation, deep tillage, and improved drainage are the most effective measures. Diagnosis of Goss's wilt is limited to recognition of variable symptoms followed by isolation onto semiselective media and identification of C. michiganensis of the subspecies that causes Goss's wilt using additional tests. Recently, Dobhal and collaborators [10] developed a loop-mediated isothermal amplification (LAMP) method to discriminate all known subspecies of C. michiganensis; however, no effective qPCR/PCR protocol is available. Although, serological and molecular diagnostic methods for economically important subspecies of C. michiganensis have been developed [2, 7, 11, 12, 13, 14] , these diagnostic tests are time consuming and may exhibit cross reactivity with Gram-positive bacteria and/or Gram-negative bacteria demanding further molecular test for cross confirmation [11, 15] .