Location: Northwest Irrigation and Soils ResearchTitle: Immunodetection of Two Curtoviruses Infecting Sugar Beet) Author
Submitted to: Plant Disease
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/8/2010
Publication Date: 7/13/2010
Publication URL: http://apsjournals.apsnet.org/doi/pdf/10.1094/PDIS-94-8-0972
Citation: Durrin, J.S., Nikolaeva, O.V., Strausbaugh, C.A., Karasev, A.V. 2010. Immunodetection of Two Curtoviruses Infecting Sugar Beet. Plant Disease. 94(8):972-976. Interpretive Summary: Curly top caused by one of three curtovirus species [Beet severe curly top virus (BSCTV), Beet mild curly top virus (BMCTV), and Beet curly top virus (BCTV)] has been a problem for the sugar beet industry as well as for the bean industry in the United States since the early 20th century. Curtoviruses are transmitted by the beet leafhopper which is widespread in semiarid areas of the western United States. Management strategies may include vector control in overwintering areas, seed- or crop-applied insecticides, and/or development of resistant cultivars. Resistant sugar beet and bean cultivars have been successfully introduced, but losses to curly top are still considerable. Maintaining resistance to curly top in sugar beet is troublesome because of yield drag and its quantitative nature. To facilitate curly top resistance breeding and possible management strategies, good detection and quantification methods are needed. In this report, we describe an enzyme-linked immunosorbent assay (ELISA) based detection protocol applicable for curly top virus detection in infected sugar beet, tomatoes, beans, and Nicotiana benthamiana, which can be scaled up for testing resistant germplasm in many breeding programs. This ELISA assay will help breeding programs select for curly top virus resistance by allowing the virus titer to be quantified.
Technical Abstract: Beet leafhopper-transmitted curly top virus is a serious problem in many different crops in the semiarid western U.S., including sugar beet, tomatoes and beans. Curly top is caused by a genetically diverse complex of phloem-limited curtoviruses. Due to the phloem restriction of curtoviruses and the lack of a convenient laboratory host-vector system for curly top virus propagation and purification, no commercial immunodetection tests are available for curtoviruses. Routine diagnostics for curly top relies either on visual symptoms or PCR tests. Lack of an ELISA test system is one of the factors hampering development and screening of the curly top resistant germplasm in, for instance, sugar beet and bean breeding programs. To fill in this gap, we developed an ELISA based detection system for curtoviruses which utilizes virus-specific antibodies generated against bacterially-expressed CP of Beet mild curly top virus. Bacterially-expressed CP was affinity purified and used as an antigen for antibody production in two animal species. Specificity of the resulting antisera was tested in Western blots and various triple-antibody sandwich (TAS)-ELISA formats with sugar beet, bean and Nicotiana benthamiana leaf tissue. We demonstrate reliable detection of two curtoviruses in different crops in TAS-ELISA format, suitable for large-scale screening of germplasm in breeding programs.