Two Components Associated with Citrus Ringspot Virus K. S. Derrick, R. H. Brlansky, R. F. Lee, L. W. Timmer, and T. K. Nguyen ABSTRACT. Infectious preparations of citrus ringspot virus (CRSV) were prepared by differen- tial centrifugation, but individual fractions following sucrose density gradient centrifugation were not infectious. Mixtures of fractions approximately 3.5 cm deep and 5.5 cm deep in the gradients were highly infectious. The two components appeared to depend on each other for infectivity. Efforts to infect citrus or herbaceous plants with single components were not successful. Index words. infectivity assays, virus purification. Citrus ringspot was first de- sucrose density gradients were done scribed by Wallace and Drake (2) in using 0.05 M Tris, 0.1% ascorbic acid, 1968. The cause of the disease has not 0.1% L-cysteine, and 0.5% 2-mercap- been determined, but it is presumed toethanol (2-ME), adjusted to pH 8.0 to be a virus and is referred to as cit- with HC1 (TACM). Young leaves of rus ringspot virus (CRSV). CRSV is Duncan grapefruit with symptoms readily transmitted to many herbace- were used in purification trials. ous plants by sap inoculation and pro- Leaves were pulverized in the pres- duces local lesions on Chenopodium ence of liquid nitrogen. The resulting quinoa, but the infectivity in extracts frozen powder was transferred to a is unstable, and there are no reports mortar and pestle at room tempera- on the physical and chemical proper- ture and ground with 2 or 7 ml of ties of the virus. Several individuals TACM per g of leaf tissue. All sub- (unpublished observations of S. M. sequent purification steps were done Garnsey, D. Gonsalves, R. F. Lee, at 4 C. The extracts were filtered and L. W. Timmer) have observed re- through cheesecloth and, in some ex- peatedly that the infectivity as- periments, stirred with Freon for one sociated with CRSV appears to be min followed by clarification by cen- lost following fractionation by sucrose trifugation for 10 min at 12,000 g. density gradient centrifugation Linear gradients of 10-40% sucrose in (SDGC). TACM were prepared in Beckman This paper shows that the infectiv- SW 41 rotor tubes. Clarified extracts ity associated with CRSV is resolved prepared in 2 ml of buffer per g of leaf into two widely separated compo- tissue were applied to gradients with- nents by SDGC. out concentration. Extracts in 7 mls per g of leaf tissue were subjected to MATERIALS AND METHODS one or more cycles of differential cen- trifugation (12,000 g for 10 min fol- Virus source. The CRSV isolate lowed by 300,000 g for 1 hr) and the used in this study was CRSV-4 that resulting pellets were suspended in had been through several successive TACM (1.0 m116 g of starting tissue). single lesion transfers on C. quinoa One ml preparations were layered followed by transfer to Gomphrena on the sucrose gradients and cen- globosa and subsequently to Duncan trifuged for 2.5 hr at 38,000 rpm at 4 grapefruit (1). C. The gradients were fractionated Partial purification. Infectivity into 0.6-ml fractions using an ISCO assays, partial purification trials and gradient fractionator. 'Florida Agricultural Experiment Stations Journal Series No. 8909. Other Virus Diseases Infectivity assay. Extracts and from SDGC of clarified extracts or fractions from gradients were as- preparations concentrated by differ- sayed for infectivity on C. quinoa. ential centrifugation were not infec- Leaves were dusted with 600 mesh tious, but some infectivity could be carborundum, and 50 p1 of extracts, recovered from a mixture of all frac- individual gradient fractions, or equal tions from the gradient. This observa- volume mixtures of two gradient frac- tion led to the finding that at least tions were spotted and rubbed onto two components that are widely sepa- leaves. The plants were maintained in rated by SDGC are required for infec- a greenhouse and lesions were tivity of CRSV. In various experi- counted 6 to 8 days after inoculation. ments, maximum infectivity was al- Assay for possible mixed infec- ways associated with mixtures of tion. Individual top and bottom gra- fractions that were approximately 3.5 dient fractions that were known to be cm and 5.5 cm deep in the gradients. infectious when mixed were used to To readily demonstrate the presence inoculate C. quinoa, G. globosa and of infectious components following Duncan grapefruit. At various times SDGC, it was necessary to perform after inoculation, extracts from these the partial purification and assay frac- plants were mixed with the appropri- tions for infectivity on the same day. ate top or bottom fractions from Partial purification by SDGC did not SDGC and used to inoculate C. stabilize the infectivity. Very little in- quinoa. fectivity could be detected when the fractions were kept on ice overnight. RESULTS AND DISCUSSION This indicates that infectivity assays cannot be used to monitor CRSV Infectivity associated with CRSV purification schemes that require has been shown to be stabilized to a more than 1 day. degree by making extracts in Tris The observation that two fractions buffer, pH 8.0 containing 0.5% 2-ME that are readily separated by SDGC (1). Unpublished studies at Lake are required for infectivity suggested Alfred have shown the addition of L- citrus ringspot may be caused by a cysteine and ascorbic acid to the Tris mixed infection of two viruses. buffer further stabilizes the infectiv- Symptoms were not observed on C. ity. Thus, TACM was used for all ex- quinoa, G. globosa, or Duncan tractions, partial purifications and in- grapefruit plants that had been inocu- fectivity trails reported here. Using lated with either top or bottom frac- Tris buffer below pH 8.0 or omitting tions. Extracts from these plants 2-ME always resulted in severe loss which had been inoculated with single of infectivity. fractions failed to produce local le- Infectivity assays on C. quinoa of sions on C. quinoa when mixed with extracts of young Duncan grapefruit a freshly prepared top or bottom frac- leaves with symptoms of CRSV (pre- tion from SDGC. These observations pared using 1 g of tissue per 10 ml of suggest CRSV is not a mixed infec- TACM) produced 80 to confluent le- tion of two competent viruses and in- sions per leaf. As previously reported dicate that the top and bottom frac- (I), most of this infectivity was lost if tions contain portions of a divided the preparation was kept on ice for 24 viral genome that depend on each hr before assay. The infectivity in ex- other for infectivity. tracts could be recovered in high- The location of the infectious com- speed pellets following differential ponents of CRSV on sucrose gra- centrifugation, but these prepara- dients should prove useful in further tions also lost most of their infectivity studies to characterize the virus and after 24 hr on ice. Individual fractions develop diagnostic tests for indexing. Tenth IOCV Conference LITERATURE CITED 1. Garnsey, S. M. and L. W. Timmer 1980. Mechanical transmissibility of citrus ringspot virus isolates from Florida, Texas, and California, p. 174-179. I n Proc. 8th Conf. IOCV. IOCV, Riverside. 2. Wallace, J. M. and R. J. Drake 1968. Citrange stunt and ringspot, two previously undescribed virus diseases of citrus, p. 177-183. I n Proc. 4th Conf. IOCV, Univ. Florida Press, Gainesville.