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

Seeds of hybrid sweet corn variety Hawaiian supersweet #10 were provided by UH seed lab, HI (Agricultural Diagnosis Service Center); seeds were sown in Sunshine#4 soil mix in 20 cm pots (1 seed/pot) -experiment was conducted in 2017 at Pope Greenhouse, College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa. Fourteen strains of C. m. subsp. nebraskensis (S1 Table) were grown on YSC medium (yeast extract 10 g/L, sucrose 20 g/L, calcium carbonate 20 g/L and agar 17 g/L) and incubated at 26°C (±2°C) for 3 days; bacterial cells were harvested by flooding each plate with 5 ml of PBS buffer (pH 7.0). Corn plants (V-3 developmental stage) were inoculated using the dropped-wounded protocol described by Ahmad and collaborators [24] with a minor modification -the third leaf of each plant was wounded half way between the sheath and the tip by making a cut across 3 veins on one side of the midrib; immediately after wounding, 10 μ l of inoculum was dropped on to the wound using a plastic dropper. Negative control plants were inoculated with 10 μ l PBS (mock inoculated). Inoculated plants were covered with a plastic bag for 24 hours to increase the humidity to facilitate infection. Leaf samples from inoculated plants were collected 30 days post inoculation (dpi); DNA was isolated from each sample using the Wizard ® Genomic DNA Purification Kit according to the manufacturer's instructions.