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United States Department of Agriculture

Agricultural Research Service

Research Project: Characterization, Epidemiology and Management Strategies of Citrus Tristeza Virus and Spiroplasma citri on Citrus in California

Location: Crop Diseases, Pests and Genetics

Title: Differentiation of citrus Hop stunt viroid variants by real-time RT-PCR and high resolution melting analysis

Authors
item Loconsole, G -
item Onelge, N -
item Yokomi, Raymond
item Abou-Kubaa, R -
item Savino, V -
item Saponari, M -

Submitted to: Conference of International Organization of Citrus Virologists
Publication Type: Abstract Only
Publication Acceptance Date: June 20, 2013
Publication Date: N/A

Technical Abstract: Viroids are small, infectious, single-stranded RNA molecules that cause several important citrus diseases. Viroids are transmitted primarily in budwood, however, spread can also occur mechanically on pruning equipment, budding knives, hedging and topping equipment. Exocortis and cachexia are two well-known viroid diseases of citrus. However, at least seven distinct viroids have now been discovered in citrus and are classified in the following groups: CVd-I, CVd-IIa,CVd-IIb, CVd-III, CVd-IV, CVd-V, and CVd-VI. Viroids can be detected by indexing on sensitive biological indicators such as Etrog citron for exocortis and group III viroids and Parson's Special mandarin for cachexia. Biological indexing requires 3-6 months for citron and at least one year for Parson's Special mandarin. There is a need, therefore, to develop a molecular test that is robust and rapid that detects and differentiates economically important citrus viroids. The RNA genome of Hop stunt viroid (HSVd) is known to contain five to six nucleotides in a variable (V) domain called the cachexia expression motif that are associated with the pathogenic and non-pathogenic citrus cachexia variants. Primers to detect several single nucleotide polymorphisms in the cachexia expression motif were developed which produced products in real-time, Reverse Transcription-polymerase chain reaction (RT-qPCR) that separated HSVd variants by High-Resolution Melting Temperature (HRM) analysis. Two independent RT-qPCR tests were developed based on different dye chemistries: (i) EVA Green (ii) TaqMan. Both tests separated HSVd variants into three distinct clusters by its unique melting temperatures with confidence level greater than 98%. Assay specificities were confirmed by nucleotide sequencing of samples within each cluster. Fifty known HSVd-infected samples from field sources from California, Italy, Spain, Syria and Turkey were accurately identified and differentiated in validation tests. Thus, rapid detection of economically important HSVd variants was achieved. The protocols can be used for rapid diagnosis of infected trees in the field and nursery and reduces the need for bioindexing and sequencing analysis.

Last Modified: 7/24/2014
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