PHYSIOLOGICAL and BIOCHEMICAL ASPECTS Localization and Detection of in Exocortis-Virus-Infected Citron

A. W. FELDMAN, R. W. HANKS, and S. M. GARNSEY

OBJECTIVESof the investigation to clone 7-4 remained symptomless, be reported in this paper were: 1, to whereas plants of all other clones determine by thin-layer chromatog- developed typical exocortis symp- raphy (TLC) the fluorescent band(s) toms. Graft-inoculated plants of OES- that best characterize exocortis virus 7 and OES-10obtained from G. D. infection in citron, and 2. to ascertain Bridges-State Department of Agri- the tissue(s1 involved and the pattern culture, Winter Haven, Florida- of distribution in citron were used in several tests. These plants after infection. The investiga- graft-inoculated plants, inoculated tion was prompted after a recent with several sources of exocortis finding that a specific violet fluores- virus, were still symptomless at cent material, gentisoyl glucose (53, time of analyses, about 7 months is present in the albedo and bark after inoculation. extract of both stubborn- and Plant tissues analyzed were: 1 greening-affected trees (8, 9), and whole tip leaves (ca. 0.5 cm long) by the more recent observation that from new shoots; 2. midribs from TLC of tissue extracts of citron clones expanding young leaves (ca. 2-3 reveal 1-3 blue to violet fluorescent cm long); 3. midribs from fully ex- bands. These bands are primarily panded young leaves; 4. midribs the coumarins, and skim- from mature leaves; 5. bark from min (Fig. 11, with moderate amounts young shoots; 6, bark from mature of both free and unidentified bound stems; 7. young root tips; 8. inactive forms of and umbel- feeder root tips; and, in some in- liferone (1). stances, 9. the woody stele from immature stems. Investigations were Methods made on tissues 1-6 beginning 3 PLANT MATERIAL.-The Sources weeks after inoculation and con- of plant material used were root- tinued for 3 months on all citron ed greenhouse-propagated cuttings clones except 7-4. With the latter from Etrog citron clones 7-4, 7-7, clone, analyses were made biweekly OES-7, OES-10, 7-26, and Arizona for 8 months. In 4 experiments, 861. A standard isolate of exocortis analyses were made on each leaf virus, free of other known viruses, midrib from the top 10 leaves, was introduced by mechanical inoc- exclusive of the very small tip leaves ulation as required (6). Plants of of new shoots, and on 8 cm strips 240 PROCEEDINGS of the IOCV of young bark taken in sequence, drolyses and two-way paper chro- beginning 3 cm below the stem tip. matography (4) in which standard These experiments and analyses curves were prepared with either were ' conducted over a 3-year scopoletin or . period and in different seasons. TISSUE EXTRACTION AND CHRO- Results MATOGRAPHY.-All tissues from TISSUES INVOLVED IN COUMARIN healthy and exocortis-infected plants ACCUMULATION AND SEQUENCE IN were finely chopped and extracted DEVELOPMENT OF FLUORESCENT at 4OC, for 72 hours, in 50 per cent BANDS. -Extracts from tip leaves, ethanol at a ratio of 1:3 w:v. Fifty to from midribs of expanding young 100 pI of tissue extract was spotted leaves, from midribs of young ex-

R = Glucose, Scopolin R = Glucose, Skimmin R = H, Scopoletin R = H, Umbelliferone FIGURE 1. Principal coumarins in extracts of midribs and young bark from Etrog citron infected with exocortis virus. on 20 x 20 cm TLC plates (Mallinck- panded leaves, from young bark, rodt 7G silica gel), which were devel- and from immature wood of all oped in H20-saturated n-butanol at exocortis-infected citron clones ex- 26 OC (1 1. After development, chro- hibited 1-3 violet to blue fluores- matograms were air-dried and cent bands on the chromatogram. viewed under uv at 366 and 253 These fluorescent bands were not- nm. with the occasional exception of tip DETERMINATIONOF TOTAL PHENOLS. leaves- present on chromatog rams -Fluorescent zones were removed from comparable tissue extracts of from the TLC plates and eluted in healthy citron (Table 1). The violet ethylacetate-acetic acid-H,O (5: 1: 1, to blue fluorescent bands were v:v), dried in vacuo at 40°C, and located at Rf 0.40-0.45, 0.47-0.59, taken up in 3 ml of 50 per cent and 0.68-0.75. When only 1 fluores- ethanol for analysis by spectro- cent band was present, it was lo- photofluorometry (SPF) (Aminco- cated at Rf 0.47-0.59. The variation Bowman spectrophotofluorometer) in the range of the Rf and the color with scopoletin as a standard. of the fluorescence of each band COUMARINDETERMINATION. -The was due to the concentration of the quantity of the specific coumarins coumarin(s) in the band and to was determined by SPF after hy- interference and accumulation of PHYSIOLOGICAL and BIOCHEMICAL ASPECTS other phenolics at the same Rf. The 0.45 in the extract from the midrib Rf for a given fluorescence was of fully expanded young leaves and always higher in extracts obtained occasionally in the young bark. This during spring and summer when band contained other bound deriva- tissues were most succulent. tives of both scopoletin and umbel- Coumarin(s) associated with the liferone, including small amounts of fluorescent band at Rf 0.47-0.59 scopolin and skimmin. Depending was always produced first, often on seasonal conditions, a third prior to visible symptoms, and fluorescent band (Rf 0.68-0.75) generally 4-6 weeks after inocula- often appeared with the second tion, depending upon seasonal fluorescent band, or 1-2 weeks conditions. This fluorescent band later.

TABLE 1. BOUND FORMS OF SCOPOLETIN AND UMBELLIFERONE IN THE PRINCIPAL BLUE TO VIOLET FLUORESCENT ZONE (RF 0.47-0.59) FROM THIN-LAYER CHROMATOGRAMS OF TISSUE FROM EXOCORTIS-INFECTED CITRON^ (AVERAGEOF 3 TRIALS) Total phenolics Bound scopoletin Bound umbelliferone ~atio:" at Rf 0.47-0.59 at Rf 0.47-0.59 at Rf 0.47-0.59 scopoletin/ pg/g tissue pg/g tissue pg/g tissue umbelliferone b Tissue (fr. wt.) (fr. wt.) (fr. wt.) Whole tip leavesd 0.95 0.34 0.12 3 Midribs from expanding top leavesd 1.65 0.68 0.28 2.4 Midribs from fully expanded young leaves 6.9 5.4 1.3 4.2 Young stem bark 12.4 8.9 1.4 6.4 Immature stem wood 11.3 5.1 0.9 5.7 Mature stem bark ~r.~ -- a. Plants of c~tronclone 7-26, sampled 2-3 months after inoculation when symptoms first appeared. b. Fluorescent zone at Rf 0.47-0.59 was not observed in the tissue extract of midribs from mature leaves, young root t~ps, and old feeder-root tips or in any other tissue extracts from healthy plants with the occasional exception of whole tip leaves. c. Concentration and ratio of bound scopoletin to bound umbelliferone will vary according to season, clone, and duration of infection. d. The fluorescent band at Rf 0.47-0.59 from these tissue extracts often was diffuse and not so well defined. e. Less than 0.1 pg/g. was found in extracts obtained The fluorescent band at Rf 0.47- either from the midribs of expand- 0.59 was found in all the citron ing young leaves, midribs of fully clones studied, whether mechani- expanded young leaves, young cally or bud inoculated, and we bark, or any 2 of these tissues or in consider it the principal fluorescent all of them. The concentration and band associated with exocortis virus ratio of bound scopoletin to bound infection in citron. This fluorescent umbelliferone in the fluorescent band, however, appeared slowly in band at Rf 0.47-0.59 varied accord- the symptomless clone no. 7-4 since ing to the tissue, duration of infection it was first detected 6 months after (Table I),season, and clone (1). inoculation in the extract of the mid- When symptoms first appeared (leaf ribs from fully expanded young epinasty), a second fluorescent leaves and 2 weeks later in the band was often observed at Rf 0.40- extract from young bark. 242 PROCEEDINGS of the IOCV Twenty-eight plants of citron the leaves that exhibited slight leaf clones OES-7 and o~s-10,which re- curl symptoms, and also in the mained symptomless 7 months after midribs of I or 2 fully expanded bud inoculation with tissue suspect- young leaves approximately 6-1 0 ed of carrying exocortis virus, were cm away but not in the midribs of examined chromatographically for the intervening leaves. the fluorescent band at Rf 0.47- 0.59. The band was found in 11 Discussion plants. These 11 and 4 others even- The accumulation of the bound tually showed visible symptoms. and free coumarins in certain tissues PATTERNOF COUMARIN ACCUMU- of Etrog citron following infection LATION IN INDIVIDUAL LEAVES AND by exocortis virus offers the possi- YOUNG BARK SEGMENTS. - Deriva- bility of using these phenolics as a tives of scopoletin and umbel- presumptive aid for detection of liferone, primarily scopolin and exocortis virus. Though bound skimmin, are always present in the forms of scopoletin and umbel- principal blue-violet fluorescent liferone are normal constituents of band at Rf 0.47-0.59. These cou- citrus (2, 3, 4) as well as of other marins are readily found in the plants, particularly those subjected extract of midribs from leaves that to stress (3,4,73, they do not appear show even very early leaf epinasty to be normal constituents of the symptoms of exocortis virus infec- citron tissues used in our tests. The tion. In some plants, only 1 leaf extraction method outlined here ap- showed early symptoms; but cou- parently removes the extracellular marins were found in the extract of or the more readily diffusable cou- the adjacent young bark or in the marins. The detection of exocortis- young bark 5-8 cm above or below specific phenolics by TLC and uv the node of the symptom-bearing is relatively simple, but careful leaf. These coumarins may also be selection of tissue is necessary to found in the extract of midribs obtain a sharp fluorescent pattern at from symptomless leaves 2-6 leaves Rf 0.47-0.59. Separately prepared above or below the leaf that exhibits extracts of midribs from fully ex- slight symptoms. When 3-4 leaves panded young leaves, and from on 1 shoot exhibit slight leaf curl young bark in an area 5-10 cm symptoms, coumarins may readily long and beginning 8 cm below the be found in extracts of midribs from shoot tip, consistently yielded the all fully expanded young leaves, best fluorescent pattern, with little with and without symptoms, and in or no interference at the principal the young bark. In some instances, Rf (0.47-0.59). Comparable tissue the distribution of coumarins was extracts from healthy citron should not uniform. Coumarins could be be included as a basis for compar- found in the midribs and young ison. bark immediately above or below Mild strains of exocortis virus may PHYSIOLOGICAL and BIOCHEMICAL ASPECTS 243 not produce symptoms in citron coumarins in midribs and young until 1 or more years after inocula- bark of citron may be caused by tion, but coumarins do accumulate erratic virus multiplication, by ir- and can be detected in the young regular or localized interference with bark and midribs from fully ex- normal host metabolism, or by both. panded young leaves prior to ap- A similar type of random distribution pearance of symptoms. Vigorous of gentisoyl glucose in the bark of growth of the host facilitates early greening-affected citrus was also detection of coumarins, and test noted by Schwarz (personal com- plants should receive good care. munication). In trifoliate orange root- A variety of stress conditions can stock infected with exocortis virus, cause increased coumarin concen- the concentration of coumarins in trations in plants. The specificity of the root bark is highest in late coumarin formation as a result of spring and early summer. Couma- exocortis infection should be re- rins are absent, or in very low con- examined whenever a stress factor centration, in fall and winter (1). that could cause coumarin accumu- This absence is in contrast to the lation is added. The effects of citrus accumulation of gentisoyl glucose in yellow vein, stubborn, and seedling- citrus affected by the viruses that yellows viruses were not investigated cause greening, dieback, and stem in this study and would require study pitting where the highest concentra- in regions where they would be tion of gentisoyl glucose is found encountered. Citron plants grown in during late fall or early winter (5, 8, metal containers may develop root 9). necrosis caused by the metal oxides Data from this investigation and of the container. Extracts of young others (1, 3, 5, 8, 9) support the bark or midribs from young ex- possibility of using specific host panded leaves from plants injured in metabolic accumulations to ascer- this fashion have shown the prin- tain the presence of a specific virus cipal fluorescence at Rf 0.47-0.59. infection in citrus. Plastic liners for cans help to elimi- ACKNOWLEDGMENTS.- The au- nate metal oxide-induced root ne- thors are indebted to C. C. Rafferty, crosis. H. Benson, and G. J. Edwards for The random accumulation of their valuable technical assistance.

Literature Cited 1. FELDMAN,A. W. 1969. Chemical changes Phenolic compounds in roots and induced in citrus plants by viruses, leaves of four citrus cultivars. Nature p. 1495-1503. In H. D. Chapman 207: 985-86. led.), Proc. 1st Intern. Citrus Symp. 3. FELDMAN,A. W., and HANKS,R. W. 1968. Vol. 3. Univ. Calif., Riverside. Identification and quantification of 2. FELDMAN,A. W., and HANKS,R. W. 1965. phenolics in the leaves and roots of 244 PROCEEDINGS of the IOCV

healthy and exocortis-infected citrus, 7. HUGHES,J. C., and SWAIN, T. 1960. p. 292-99. In J. F. L. Childs (ed.), Proc. Scopolin production in potato tubers 4th Conf. Intern. Organization Citrus infected with Phytophthora infestans. Virol. Univ. Florida Press, Gainesville. Phytopathology 50: 398-400. 4. FELDMAN,A. W., and HANKS,R. W. 1968. 8. SCHWARZ,R. E. 1968. Thin layer chro- Phenolic content in the roots and matographic studies on phenolic leaves of tolerant and susceptible citrus markers of the greening virus in cultivars attacked by Radopholus simi- various citrus species. S. African J. lis. Phytochemistry 7: 5-1 2. Agr. Sci. 11 : 797-802. 5. FELDMAN,A. W., and HANKS,R. W. 1969. 9. SCHWARZ,R. E. 1969. Indexing of green- The occurrence of a gentistic glucoside ing and exocortis through fluorescent in the bark and albedo of virus-infected marker substances, p. 1 18-24. In J. F. citrus trees. Phytopathology 59: 603-6. L. Childs (ed.), Proc. 4th Conf. Intern. 6. GARNSEY,S. M. 1967. Exocortis virus of Organization Citrus Virol. Univ. Florida citrus can be spread by contaminated Press, Gainesville. tools. Proc. Florida State Hort. Soc. 80: 68-73.