UC Riverside International Organization of Citrus Virologists Conference Proceedings ( 1957-2010 ) Title Replication / Accumulation and Symptom Expression of Citrus Viroids on Some Species of Citrus and Related Genera Permalink

Plants of 32 species of Citrus and related genera grafted on rough lemon rootstock were inoculated with an artificial mixture of citrus viroids containing Citrus exocortis viroid , Citrus viroid I, Citrus viroid II, Citrus viroid III and Citrus viroid IV . Infection and viroid titers were determined by sPAGE and molecular hybridization analysis. Plants in which viroid infection could not be detected were further indexed by inoculation on Etrog citron 861 S1. Under the conditions of this assay most of the species were symptomless carriers. Only Citrus excelsa, C. ichangensis, C. karna, C. latifolia , C. meyeri and C. pyriformis developed symptoms as a result of viroid infection. Comparative analysis of nucleic acid extracts from bark versus leaf blade tissues indicated that in 10 species, viroid that were readily detectable from bark were undetectable from leaf blade tissues by sPAGE. Citrus can be infected by several viroids (7) including Citrus exocortis viroid (CEVd) and specific variants of Citrus viroid II (CVd-II, Hop stunt viroid ) that cause the exocortis and cachexia diseases respectively (17, 24, 25). Most viroid-host combinations appear to be symptomless, and exocortis and cachexia symptoms are only observed when their viroid agents infect sensitive species. Trifoliate orange, its hybrids (the citranges) and Rangpur lime, used as rootstocks, and the Etrog citron indicator have been described as exocortis sensitive species. Alemow, Rangpur lime and Palestine sweet lime used as rootstocks, and several mandarins, including clementines, satsumas and their hybrids are sensitive to cachexia. Reports dealing with the expression of exocortis and cachexia symptoms on sweet limes (23), citrumelo (3) and Cleopatra mandarin (13) were based on the observation of symptoms on plants infected with field isolates characterized only by the response of exocortis and cachexia indicators. At present, the effect of specific viroids species has only been tested on Etrog citron (7) and trifoliate orange (21), whereas the response of most species of Citrus and related genera to viroid infection remains unknown. The objective of this study was to evaluate the response of 32 accessions of the Instituto Valenciano de Investigaciones Agrarias (IVIA) germplasm bank to viroid infection. Such a wide-ranging study has not been previously reported. MATERIALS AND METHODS Plant materials and viroid inoculation. Plants of 22 species of Citrus and 10 species of related genera (Table 1) of the IVIA germplasm collection (www.ivia.es) grafted on rough lemon rootstock were barkgraft inoculated with an artificial mixture of citrus viroids maintained in Fino lemon. The inoculated plants were kept under greenhouse conditions at 28-32°C for 5 yr before initiating this study. The mixture of citrus viroids used as inoculum had been previously obtained by graft inoculation of Fino lemon plants with the following viroid sources: CEVd (E-117) (9), Citrus viroid I (CVd-I, Citrus bent leaf viroid ) (variant Ia) (8), Citrus viroid II (CVd-II, Hop stunt viroid ) (IIa-117 and X-707) (14), Citrus viroid III (variant IIIb) (8), and Citrus viroid IV (CVd-IV) (16). In all instances viroid infection was determined by sPAGE and F iteen th IO C V C on fren e, 202— V ioid s 65 TABLE 1 DETECTION OF CITRUS VIROIDS ON INOCULATED SPECIES OF CITRUS AND CITRUS RELATIVES Citrus and citrus relatives Accession number Viroid detection and titer z Symptoms CEVd CVd-I CVd-II CVd-III CVd-IV Citrus bergamia Risso and Poit. (cv. Calabria) IVIA–254 4 2 3 3 4 — Citrus depressa Hay IVIA–238 4 3 2 4 3 — Citrus excelsa Wester IVIA-167 1 3 2 4 4 + Citrus grandis L. Osb. IVIA-321 1 1 2 2 1 — Citrus halimii B.C. Stone IVIA-278 1 2 1 4 4 — Citrus hystrix DC. IVIA-178 2 0 1 2 2 — Citrus ichangensis Swing. IVIA-235 2 2 0 3 3 + Citrus karna Raf. IVIA-242 4 4 3 4 3 + Citrus latifolia Tan. (cv Bearrs) IVIA-124 1 0 1 4 4 + Citrus limon L. Burm f. (cv. Verna) IVIA-50 4 2 2 3 2 — Citrus macroptera Montr. IVIA-279 1 2 0 3 2 — Citrus madurensis Lour. IVIA135 4 1 2 4 4 — Citrus meyeri Y. Tan. (cv. Meyer) IVIA-145 0 2 2 4 4 + Citrus myrtifolia Raf. (Hoja G) IVIA-136 1 3 3 4 3 — Citrus myrtifolia Raf. (Hoja P) IVIA-137 1 2 3 4 4 — Citrus pyriformis Hassk. (cv. Ponderosa) IVIA-268 3 1 3 3 3 + Citrus shunkokan Hort. ex Tan. IVIA-241 0 1 0 4 0 — Citrus sunki Hort. ex Tan. IVIA239 1 2 2 2 2 — Citrus tachibana (Mak.) Tan. IVIA-237 1 1 2 2 1 — Citrus temple Hort. ex Y.Tan. IVIA-81 3 3 2 4 4 — Citrus unshiu (Mak.) Marc. (cv. Clausellina) IVIA-19 0 1 1 1 0 — Citrus webberi Wester IVIA-234 1 2 2 3 3 — Atalantia citroides Pierre ex Guill IVIA-180 0 0 0 0 0 — Fortunella crassifolia Swing. IVIA-280 1 0 1 2 1 — Fortunella margarita Lour. Swing. IVIA-138 0 2 3 4 4 — z Viroids were detected by sPAGE and molecular hybridization analysis of bark tissue samples. Viroid titers were rated accordingly with the intensity of the sPAGE bands and the hybridization signal: (0) not detected, (1) sPAGE negative and very weak hybridization signal, (2) sPAGE positive and weak hybridization signal, (3) sPAGE positive and intense hybridization signal, (4) sPAGE positive and very intense hybridization signal.