Procedures and Results

PROCEEDINGS of the IOCV Further Studies on the Complex Causing Likubin of Citrus in Taiwan H. J. SU and T. MATSUMOTO

LIKUBINhas been prevalent in Taiwan component and to clarify the nature since 1958, attacking most of the of the complex. commercial varieties such as ponkan, tankan, and sweet orange. Procedures and Results Formerly thought to be due to un- ELECTRONMICROSCOPY.- Leaf dip favorable soil conditions, such as preparations derived from West In- high moisture content and deficiency dian lime, ponkan, and Eureka lemon of essential nutrients, or to root rot, plants infected by means of aphids it was subsequently demonstrated to that had fed on a source of the liku- be caused by an infectious agent be- bin complex (LKC) were negatively lieved to be closely related to tristeza stained and examined in an electron virus because it induced the tristeza microscope. The preparations were reaction in West Indian lime and Eure- seen to contain flexuous rods ap- ka lemon and was transmitted by the proximately 2000 nm long, corres- tropical citrus aphid Toxoptera citri- ponding in size and shape with cidus Kirk. (9, 10). The tristeza virus those of tristeza virus described by from likubin was, however, found to Kitajima et al. (5, 6, 16) and by Price differ somewhat from that in other (1 2). It is concluded that tristeza countries; it affected ponkan rooted virus definitely is present in likubin- on sunki, Cleopatra mandarin, and infected tissue in Taiwan. Troyercitrange rootstocks, which are COMPARISONOF LKC WITH TRISTEZA reported to be resistant to tristeza VIRUS ISOLATED BY INSECT VECTORS virus in North and South America. OR FILTER PLANTS.- Indexing some Aeglopsis chevalier; also indexed healthy looking ponkan, tankan, and positive with the virus. Moreover, sweet orange trees growing on sunki positive reactions in lime seedlings rootstocks resulted in severe tristeza were obtained by grafting them with reactions in the lime index plants. scions from healthy looking ponkan From this result, it was concluded and tankan trees on sunki rootstock. that one or more pathogens other Because of these discrepancies, it than tristeza virus are involved in was assumed that some unknown LKC. Experiments were then made component, in addition to tristeza vi- to determine whether certain com- rus, was associated with likubin. This ponents of the LKC could be filtered paper reports the results of investi- out by certain citrus indicator plants gations made to identify the unknown and by aphid and psyllid vectors. In STUBBORN and RELATED DISEASES 29 a preliminary test, tristeza virus sepa- rooted on sunki than the original rated from LKC by filtration through LKC maintained in ponkan seedlings Mexican lime or transmission by the (Fig. 1 ,A,B). All inoculum sources tropical citrus aphid was less viru- derived from Eureka lemon, sunki, lent in plants of ponkan and tankan Rangpur lime, sweet orange, pon-

FIGURE 1. comparative response of citrus plants to likubin complex (right) and to tristeza virus (left). A Ponkan on sunki rootstock. 13. Tankan on sunki. C. Szinkom seedlings. D. Cala- mandarin seedlings. E. Likubin-diseased leaf of Valencia sweet orange showing a chlorotic pattern with dark green spotting. PROCEEDINGS of the IOCV kan, and tankan seedlings used as orange, Rangpur lime, Etrog citron, test filter plants produced severe ponkan, tankan, sunki, Szinkom, and symptoms in the test stock-scion calamandarin seedlings and to pon- combinations similar to those in- kan and tankan scions on sunki root- duced by the original LKC. Presum- stock. The original LKC produced ably these plants did not filter out the typical likubin symptoms in ponkan main component of LKC. Further and tankan grafted on sunki, chlo- experiments were made with the rotic patterns like those of nutritional original LKC and its tristeza virus deficiencies, and dark green spotting component. The results are sum- on leavesof Valencia orange-similar marized in Table 1 and Figures to the symptoms (Fig. 1,E) caused by 1 and 2. greening disease (8, 1 1)-and chlo- The tristeza component and LKC rotic patterns on leaves of calaman- produced similar symptoms in Mexi- darin and szinkom mandarin (Fig. can lime, Eureka lemon, and grape- 2,D,E)-like those induced by leaf fruit plants; they seemed to be equally mottling in the Philippines (1 3). The virulent in these cultivars. On the con- symptoms induced by LKC on sweet trary, no visible symptoms were pro- orangewerealso very similar to those duced by the tristeza virus compo- of stubborn reported from the United nent transmitted by means of aphids States (1). These results lead to the or by Mexican lime grafts to sweet conclusion that an unknown patho-

TABLE 1. COMPARATIVERESPONSES OF VARIOUS INDICATOR PLANTS TO INOCULATION WITH THE LIKUBIN COMPLEX BY MEANS OF GRAFTS AND TRISTEZA VIRUS BY MEANS OF APHIDS OR BY MEANS OF GRAFTING WITH SCIONS FROM MEXICANLIME PLANTS Indicator ~lant Likubin com~lex(LKC) Tristeza-virus comoonent Mexican lime Vein clearing and corking; leaf flecking, cupping, and Same as with LKC atrophy; stem pitting; stunting Eureka lemon Yellowing, chlorosis, and atrophy of leaf; stunting Same as with LKC Grapefruit Chlorosis, dark green spotting on leaves; cupping and Chlorosis, cupping, and atrophy atrophy of leaf; stunting of leaf, stunting Sweet orange Chlorotic pattern of zinc deficiency, and dark green spot- No visible symptom (Valencia) ting on leaf; atrophy of leaf; stunting Rangpur lime Yellow mottling of leaf; stem pitting; phloroglucinol test No visible external symptom ex- negative cept stem pitting Etrog citron Yellow mottling of leaf; stem pitting No visible external symptom ex- (Arizona 861 or cept stem pitting USDCS 60-13) Szinkom Chlorotic pattern of zinc deficiency, atrophy of leaf; No visible symptom mandarin stunting Calamandarin Chlorotic pattern of zinc deficiency, atrophy of leaf; No visible symptom stunting Vein yellowing, swelling and corking; leaf yellowing or No striking symptom; loss of chlorosis; rigid; premature defoliat~on;dieback vigor Symptoms similar to those in ponkan/sunki, in addition No visible symptom to mild stem pitting Sunki Leaf yellowing with deficiency patterns, vein swelling, No visible symptom and corking STUBBORN and RELATED DISEASES 3 1 gen, in addition to tristeza virus, is passage through Mexican lime seed- involved in likubin disease and that lings. this unknown component may be the IDENTIFICATION OF THE UNKNOWN main pathogen of this disease. Evi- COMPONENT OF LKC.-Plants infected dently it was filtered out by passage of with LKC were indexed into seedlings LKC throug h the aphid vectors and by of cultivars regarded as indicator

FIGURE 2. Symptoms of likubin in leaves of various citrus cultivars-symptoms consisting of prominent and corky veins, chlorotic patterns resembling those of nutritional deficiencies, and dark green spotting. A. Ponkan. 8. Tankan. C. Sunki. D. Szinkom mandarin. E. Calaman- darin. 3 2 PROCEEDINGS of the IOCV plants for certain citrus viruses, as seedlings inoculated with LKC did follows. not develop the young-leaf symp- A. Tests for exocortis virus. - Pon- toms (vein flecking and zonate oak- kan, tan kan, and sweet orange scions leaf patterns) of psorosis A and con- carrying LKC were grown on root- cave gum. stocks of trifoliate orange and Rang- E. Tests for cachexia-xyloporosis. pur lime. No bark scaling or yellow -No gummy pegs on the bark or blotching was noticed on the root- small pits in the wood were found in stocks although the tops were yel- Rangpur lime stock topworked with lowed and stunted. Staining the med- likubin-diseased ponkan, although ullary ray cells of bark from Rangpur some groove-shaped pits did form in lime, trifoliate orange, ponkan, tan- the stock wood. kan, sweetorange, and Mexican lime F. Tests for stubborn, greening, plants carrying LKC with phloro- and leaf-mottling pathogens. - LKC glucinol gave negative results. Etrog caused mottled-leaf patterns that re- citron (Arizona 861 and us~cs60- sembled nutritional disorders. Other 13) plants grafted with budwood of symptoms included dark green ponkan, Rangpur lime, and Mexican spotting in yellowed leaves, yellow limeplantscarrying LKC did not devel- vein banding, thick leaves with prom- op epinasty of leaves or cracking of inent veins, stunting, and twig die- the underside of the midveins. The back in sweet orange seedlings citron plants did, however, develop (Valencia and Leuchen) and resem- yellow mottled leaves and stem pit- bled those of stubborn and greening ting. It isconcluded that the unknown diseases described by Calavan and component of LKC is not exocortis Christiansen (2) and Schwarz (14) virus. (Fig. 1,El. The similarity of likubin B. Tests for satsuma dwarf virus. - and leaf-mottling diseases was also Seedlings of blackeye cowpea and noted in the symptoms (chlorotic white sesame, kindly supplied by Dr. leaf patterns resembling zinc-defi- S. Yamada, gave a negative response ciency patterns and atrophy of to mechanical inoculation with LKC, leaves and shoots) in the graft- indicating the absence of satsuma inoculated Szinkom and calaman- dwarf virus in the inoculum (1 7). darin seedlings, provided by the C. Tests for citrus-variegation and courtesy of Mr. C. I. Gonzales (Fig. crinkly-leaf viruses. -Seedlings of 2,D,E). Attempts to transmit with blackeye cowpea inoculated with the citrus psyllid (Diaphorina citri LKC developed no chlorotic local Kuw.) have been made since the fall lesions indicative of these viruses (4). of 1968, but no positive results have Leaves of inoculated Eureka lemon been obtained. Trials are still in seedlings developed no pinpoint progress. Cucumber seedlings in- spotting. oculated with the likubin pathogen D. Tests for concave-gum and did not develop symptoms. Chro- ~sorosis viruses. -Sweet orange matographic analyses of albedo and STUBBORN and RELATED DISEASES bark extracts from likubin-diseased thevirus. Theeffect of tristezavirus on ponkan, tankan, and sweet orange the development of likubin is still un- plants were made by the methods determined because isolates of the described by Schwarz (15) for de- unknown component free of tristeza tecting the fluorescent marker sub- virus are still unavailable. Neverthe- stance considered to be specific for less, it is apparent that tristeza virus the greening disease. Bright violet- does not add materially to the severity blue spots were barely visible in the of the disease because in a prelimi- chromatic profile at Rf 0.1 under nary experiment inoculum buds uv light. from trifoliate orange plants-which are immune from tristeza virus- Discussion and Conclusions graft-inoculated with LKC, recently induced likubin symptoms as severe In previous reports (9, 10) it was as those caused by the original suggested that likubin is caused by LKC a virus or viruses closely related to Calavan (1) suggested that likubin that of tristeza. It is now apparent that in Taiwan and yellow shoot in main- an unknown component is associ- land China (3,7)are similar to green- ated with this disease and that this ing and stubborn in many respects. unknown component may be the In view of this similarity, it is likely main component of the causal com- that the main component of LKC is a plex. Tristeza virus is present even in pathogen closely related to those healthy looking trees in Taiwan. that cause stubborn, greening, and Moreover, tristeza virus isolates from leaf-mottling diseases. Nevertheless, LKC did not produce likubin symp- chromatographic analyses of the toms in commercial stock-scion albedo and the bark of likubin- combinations whereas grafts from affected trees have failed to give a likubin-affected trees caused typical positive fluorescent test, and at- likubin symptoms. tempts to transmit the disease by Latent infection by tristeza virus is means of the citrus psylla have been widespread in Taiwan in ponkan, negative. Thus, it is too early to con- tan kan, Leuchen, and Valencia sweet clude that the likubin pathogen is orange trees on sunki rootstock, but related to the greening pathogen or these combinations are tolerant of to the stubborn pathogen. Ire Cited 1. CALAVAN,E. C. 1968. A review of stubborn toms of stubborn disease, p. 69-76. In and greening diseasesof citrus, p. 105- W. C. Price(ed.1, Proc. 2nd Conf. Intern. 17. In J. F. L. Childs (ed.), Proc. 4th Organization Citrus Virol. Univ. Florida Conf. Intern. Organization Citrus Virol. Press, Gainesville. Univ. Florida Press, Gainesville. 3. FOAN,H. C., and LIU, C. C. 1965. The in- 2. CALAVAN,E. C., and CHRISTIANSEN,D. W. sect vector and indicator plants of the 1961. Stunting and chlorosisinduced in citrus yellow-shoot disease. Acta Phy- young-line citrus plants by inoculations tophylac. Sin. 4: 219-24. (RAM 45: from navel orange trees having symp- 201, 1966.) PROCEEDINGS of the IOCV

4. GRANT,T. J., and CORBETT,M. K. 1960. 12. PRICE, W. C. 1966. Flexuous rods in Mechanical transmission of the infec- phloem cells of lime plants infected tiou; ~ariegationvirus of citrus. Nature with citrus tristeza virus. Virology 29: 188:51 9-20. 285-94. 5. KITAJIMA, E. W. et al. 1963. Thread-like 13. SALIBE,A. A., and CORTEZ,R. E. 1968. particle associated with tristeza disease Leaf mottling-a serious virus disease of citrus. Nature 201 : 101 1-1 2. of citrus in the Philippines, p. 131-36. In 6. KITAJIMA,E. W. et a!. 1965. Electron micro- J. F. L. Childs (ed.), Proc. 4th Conf. In- scopical ~nvestigation on tristeza, p. tern. Organization Citrus Virol. Univ. 1-9. In W. C. Price (ed.), Proc. 3d Florida Press, Gainesville. Conf. Intern. Organization Citrus Virol. 14.SCHWARZ, R. E. 1964. An insect-transmis- Univ. Florida Press, Gainesville. sible virus, trapped on sweet orange 7. LIN. K. H. 1964. Remarks on notes on citrus seedlings in orchards where greening yellow shoot disease. Acta Phytophylac. disease is common. S. African J. Agr. Sin. 3: 163-71. (RAM 44: 205, 1965.) Sci. 7: 885-90. 8. MCCLEAN,A. P. D., and OBERHOLZER, 15. SCHWARZ,R. E. 1968. The mechanical P. C. J. 1965. Greening disease of transmission of the greening virus to the sweet orange: Evidence that it is cucumber, p. 264-66. In J. F. L. Childs caused by a transmissible virus. S. (ed.), Proc. 4th Conf. Intern. Organiza- African J. Agr. Sci. 8: 253-76. tion Citrus Virol. Univ. Florida Press, 9. M~~su~o~o,T.,andSu,H. J. 1966. Likubin Gainesville. or huanglungpin, citrus virus disease 16. SILVA,D. M., OLIVEIRA,A. R., and KITA- closely related to tristeza (I). J. Agr. JIMA. E. W. 1965. Partial purification of Assoc. China, New Ser. No. 56: 1-1 2. tristeza virus, p. 10-1 3. In W. C. Price 10. MATSUMOTO,T., WANG,M. C., and Su, H. led.), Proc. 3d Conf. Intern. Organiza- J. 1961. Studies on likubin, p. 121-25. tion Citrus Virol. Univ. Florida Press, In W. C. Price (ed.), Proc. 2nd Conf. Gainesville. Intern. Organization Citrus Virol. Univ. 17. TANAKA,S., and KISHI, K. 1963. Studies Florida Press, Gainesville. on indicator plants for citrus viruses. 11. OBERHOLZER,P. C. J. et al. 1965. Green- 1. Mechanical inoculation on legumi- ing disease of sweet orange in South nous plants with sap from Satsuma Africa, p. 21 3-1 9. In W. C. Price (ed.), dwarf tree. Ann. Phytopathol. Soc. Proc. 3d Conf. Intern. Organization Cit- Ja~an28: 262-69. rus Virol. Univ. Florida Press, Gaines- ville.