Citrus Virologists Conference Proceedings ( 1957-2010 ) Title A Mild Isolate of Citrus Variegation Virus Found in Florida Citrus Permalink

Random indexing of symptomless rough lemon in a recently replanted grove site near Dundee, Florida, revealed a mechanically transmissible virus which produced mild leaf mottle symptoms on Etrog citron. Host-range studies, serological tests, and physical properties of purified virus indicated that the virus was an isolate of citrus variegation virus (CVV). This newly discovered CVV isolate produced significantly milder symptoms in diagnostic indicators than the previously described Florida isolate of CVV and was more difficult to detect in graft-inoculated indicators. An antiserum was prepared against the new CVV isolate and used for serological assays. Survey of the discovery site by enzyme-linked immunosorbent assay (ELISA) indexing revealed CVV-infected sprouts only in a limited area. Index words. Crinkly leaf, citrus leaf rugose virus. Citrus variegation virus (CVV) has been found in several citrusgrowing areas including Florida (4, 6, 10) ; however, i t is usually not widespread or a major disease problem. The isolate of CVV originally described from Florida (6) was considered a typical isolate, and causes strong symptoms in Eureka lemon and Etrog citron. We had assumed that other CVV isolates would be readily detected during routine indexing for citrus exocortis viroid (CEV) on Etrog citron. A citrus crinkly leaf virus (CCLV) has also been reported in citrus from several locations (4, 5, 10, 12). Symptoms of CCLV on lemon are milder than those described for CVV and consist of chlorotic flecking and mild distortion. A close relationship between CVV and CCLV was suggested when they were first described (4) and later substantiated by further study (2, 10, 12, 13). A crinkly leaf-type virus (CLTV) was described from Florida which 'Mention of a trademark, warranty, proprietary product, or vendor does not constitute a guarantee by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable. produced flecking symptoms similar to CCLV in lemon (8). Subsequent studies showed that CLTV was clearly distinguishable from CVV in biological, physical, and serological properties, and the name citrus leaf rugose virus (CLRV) was proposed (8) and adopted (9) . Recently, we discovered by chance an isolate of CVV in Florida, which produced only mild symptoms in Etrog citron and which, under warm conditions, was difficult to detect by symptoms. This paper describes the discovery of this mild isolate of CVV, its symptoms in citrus and noncitrus plants, and a survey for its incidence in the discovery location. A companion paper (3) describes purification of the mild isolate, some of its physical properties, and development of a specific antiserum. METHODS AND MATERIALS Plant materials and growing conditi~ns. Most indexing work was done in a partially shaded glasshouse cooled by evaporative coolers. Assays on herbaceous plants were made in spring when temperatures normally ranged from 20 to 26°C. Indexing on citrus indicators and observation of sympOther Graf t-Transmissible Agents toms were done all year. In summer, glasshouse temperatures frequently reached 30 to 32°C. Herbaceous plants were frequently grown prior to inoculation under supplemental light provided by Sylvania Gro lux@ wide spectrum VHO fluorescent tubes. The combination of natural and supplemental light yielded rapidly-growing, succulent plants suitable for sap inoculation studies. Plants were held under natural light following inoculation. All plants were grown in a steam-sterilized potting medium and were fertilized periodically to maintain good growth. Plants were grown from seed, except Eureka lemon and Etrog citron which were propagated as rooted cuttings. Virus isolates. Two isolates of CVV were used in this study. One was a previously described isolate of CVV (6, 7) originally discovered by Grant and Smith (6). A culture of this isolate is now deposited with the American Type Culture Collection as PV 196. We used a singlelesion culture (designated CVV-1) which has been passed repeatedly between citrus and herbaceous hosts and maintained for over 12 years with no apparent change. The second isolate of CVV was the mild isolate described in the introduction which was coded CVV2. Unless otherwise noted, a singlelesion isolate from Crotalaria spectabilis, which was propagated in citron and coded CVV-BA, was used for all comparative studies. Mechanically transmitted, pure cultures of CEV, CLRV, citrus ringspot virus (CRSV), tat ter leafcitrange stunt virus (TL-CSV), citrus tristeza virus (CTV) were used for the comparative tests. A psorosis isolate free of other known viruses was also used. Indexing. Citrus plants were graft-inoculated with leaf pieces or bark chips, and the plants topped to force new growth. Herbaceous plants were inoculated mechanically as previously described (6). Young leaf tissue was triturated in 10 parts 0.05 M Tris (Tris-hydroxy amino methane) pH 7.8 or 0.05 M potassium phosphate pH 7.0, with or without 0.5% 2mercaptoethanol. Citrus plants were inoculated mechanically using the same procedure on both surfaces of young leaves. Infection in symptomless hosts was confirmed by assays to cowpea or by serological tests. Serology. Serological tests were done by agar gel double diffusion methods as described previously (7, 8) or by double sandwich enzyme-linked immunosorbent assay (ELISA) . The ELISA procedures used were essentially those used for citrus tristeza virus ( I ) , except that test samples were normally prepared in 0.05 M Tris buffer. Production of the antiserum, yglobulin purification and enzyme conjugation are described else-