Two Components Associated with C

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					                   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(unpublished observations of S. M.                     sequent purification steps were done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.