Comparison of Different Diagnostic Methods for Detection of Hop stunt viroid and Citrus exocortis viroid in Citrus

Abstract

Viroids such as Hop stunt viroid (HSVd) and Citrus exocortis viroid (CEVd) are important pathogens in citrus plants. A comparative assay was performed to evaluate the viroid diagnostic techniques currently available at the Citrus Sanitation Center of Estacion Experimental Agroindustrial Obispo Colombres. We compared the results obtained in the diagnosis of HSVd and CEVd in citrus using two molecular techniques: s-PAGE and RT-PCR and biological indexing. Biological indexing was performed in a greenhouse under controlled temperature, using Etrog citron Arizona 861-S1 grafted on rough lemon rootstock as the indicator plant. Tissue samples for s-PAGE were obtained from inoculated citrons used in the biological indexing tests, and DNA extraction was performed following published procedures. HSVd and CEVd specific primers were used for RT-PCR, and samples were taken directly from field plants and inoculated citrons. Two nucleic acid extraction methods were compared. A total of 12 viroid isolates collected from orange, citron, grapefruit, citrumelo and lime trees, mostly from North Western Argentina, were used in these assays. The three diagnostic methods compared gave similar results in 11 of 12 samples assayed. Several viroids were detected by s-PAGE and most of them were found as mixtures. HSVd and CEVd were identified by RT-PCR and no differences were found between the two extraction methods assayed. Sample R-407 was positive for exocortis with biological diagnosis and s-PAGE, but it was negative with RT-PCR. This was probably due to inefficient cDNA synthesis or to the presence of inhibitors that could interfere with the reaction. On the basis of the results obtained, it can be concluded that the three techniques assayed are complementary and useful tools for diagnosis and identification of HSVd and CEVd in citrus.