Inoculation and detection of Impatiens necrotic spot virus in tomato and cucumber Objectives Kristin Vickers, Dr. Linda Hanley-Bowdoin, Mauricio Montero,and Natalia Barboza Vargas. 1 North Carolina State University, Raleigh, NC and 2 University of Costa Rica, San Jose, Costa Rica. Introduction There were two positives as a result from the spectrophotometer, which were labled as T152t.b and T154p.b (Table 1). ** Presented at the: 22 nd Annual Undergraduate Research Symposium, North Carolina State University, July 2013, Raleigh, NC. Materials and Methods Table 1. Final ELISA results for tomato and cucumber. T Results and Discussion The overall goals of this project were : 1)To inoculate economically or experimentally important plant species with a set of INSV isolates for their biological characterization. 2)To confirm mechanical inoculation results by means of ELISA (enzyme-linked immunosorbent assay). 3)Get to know the techniques for data analysis and presentation. Inoculation 7 INSV samples to inoculate with: T101, T107, T152, T154, T156 China (C+), Buffer (C-) Selected 42 plants to inoculate 21 tomato 21 cucumber Ground tissue from samples with 3% NaSO 3 with a mortar and pestle Apply solution onto selected plants Spray with carborundum Rub final layer of liquid on plants Tissue Collection Collected samples from each plant Ground tissue into liquid to be placed in microtubes ELISA Day 1 Make a solution of antibody and carbonate buffer Add 100µL to each well Day 2 Clean plates by emptying and filling with wash buffer Fill with 100µL of sample This project was funded by the National Science Foundation, as part of the IRES (International Research Experiences for Students) program. Future Work The low positives could have been caused by inoculation, frozen samples used, rejection by defense system, or time constraint. I would like to have performed the ELISA process again after one more week with hopes of higher number of positive results. Acknowledgements SampleResults+/- T101t.a T101t.b T101t.c0.04- T107t.a T107t.b T107t.c T152t.a T152t.b T152t.c T154t.a T154t.b T154t.c T156t.a0.05- T156t.b T156t.c C-t.a C-t.b C-t.c C+t.a C+t.b C C C SampleResults+/- T101p.a T101p.b T101p.c T107p.a T107p.b T107p.c T152p.a T152p.b T152p.c T154p.a T154p.b T154p.c0.05- T156p.a0.04- T156p.b T156p.c C-p.a C-p.b C-p.c C+p.a C+p.b C C C All of the species within the Tospovirus genus are transmitted by ten different species of thrips through a circulative and propagative manner. These viruses must be presented to the first or second larval stages for the adult thrip to emerge and transmit the virus, providing a “dead-end” epidemiology. Visual symptoms include necrosis, spotting, rings forming, lesions, yellow pale streaks, and reduction in yield and plant size (Pappu, 2009). Impatiens necrotic spot virus (INSV) is part of the genus Tospovirus and the family Bunyaviridae. This virus is one of the most devastating to many important vegetables, legumes, and ornamental plants throughout the world, particularly in Asia and South America (Mandal, 2012). Nineteen species of Tospovirus have been discovered up to this point. Diagnosing this virus can be done using a common serological detection test, enzyme-linked immunosorbent assay (ELISA). The Sandwich ELISA virus detection test uses an antibody/enzyme combination to trap the specific antigens of a specific virus with color change to determine if positive for that virus (Clark, 1977). Day 3 Clean with wash buffer Add conjugate antibody to wells Incubate Substrate is added Run plate in spectrophotometer