Immunodetection of Two Curtoviruses Infecting Sugar Beet

Abstract

Beet leafhopper-transmitted curly top virus is a serious problem in many different crops in the semiarid western United States, 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 rely either on visual symptoms or on polymerase chain reaction (PCR) tests. Lack of an enzyme-linked immunosorbent assay (ELISA) 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 capsid protein (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.