Rapid differentiation of citrus Hop stunt viroid variants by real-time RT-PCR and high resolution melting analysis

PubMedID: 23932930 The RNA genome of pathogenic and non-pathogenic variants of citrus Hop stunt viroid (HSVd) differ by five to six nucleotides located within the variable (V) domain referred to as the "cachexia expression motif". Sensitive hosts such as mandarin and its hybrids are seriously affected by cachexia disease. Current methods to differentiate HSVd variants rely on lengthy greenhouse biological indexing on Parson's Special mandarin and/or direct nucleotide sequence analysis of amplicons from RT-PCR of HSVd-infected plants. Two independent high throughput assays to segregate HSVd variants by real-time RT-PCR and High-Resolution Melting Temperature (HRM) analysis were developed: one based on EVAGreen dye; the other based on TaqMan probes. Primers for both assays targeted three differentiating nucleotides in the V domain which separated HSVd variants into three clusters by distinct melting temperatures with a confidence level higher than 98%. The accuracy of the HRM assays were validated by nucleotide sequencing of representative samples within each HRM cluster and by testing 45 HSVd-infected field trees from California, Italy, Spain, Syria and Turkey. To our knowledge, this is the first report of a rapid and sensitive approach to detect and differentiate HSVd variants associated with different biological behaviors. Although, HSVd is found in several crops including citrus, cachexia variants are restricted to some citrus-growing areas, particularly the Mediterranean Region. Rapid diagnosis for cachexia and non-cachexia variants is, thus, important for the management of HSVd in citrus and reduces the need for bioindexing and sequencing analysis. © 2013 Elsevier Ltd. National Research Council California Department of Food and Agriculture Regione Puglia: SCB 10005 Some of the equipment and partial support for this work was provided by Regione Puglia – Rete di Laboratori Pubblici SELGE, Specialty Crops Block Grant SCB 10005 from the California Department of Food and Agriculture and from the Italian Ministry of Economy and Finance to the National Research Council for the project “Innovazione e Sviluppo del Mezzogiorno–Conoscenze Integrate per Sostenibilità ed Innovazione del Made in Italy Agroalimentare–Legge n.191/2009”. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. Appendix A