Transmission of Citrus Tristeza Virus (CTV) from Trifoliate Orange

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Transmission of Citrus Tristeza Virus (CTV) from "Declinamiento"-Diseased Sweet Orange Trees on Trifoliate Orange Rootstocks and its ~pidemiologyin Relation to the Flight Activity of the Vector, Toxoptera citricida Kirk. (Homoptera: Aphididae) in Misioness, Argentina1 0. Nickel, F. Klingauf, and H. U. Fischer

ABSTRACT. Declining and healthy sweet orange trees on trifoliate orange rootstock in Misiones, Argentina, were shown to be infected with citrus tristeza virus (CTV). The virus was acquired readily by the tropical citrus aphid (T. citricida) and trans- mitted to three test plants: a) Key lime seedlings, b) 'Calder6n' sweet orange on trifoliate orange, and c) 'Koethen' sweet orange on trifoliate orange. Trifoliate orange seedlings were also inoculated, but could not be infected. Transmission trials were carried out with single aphids and with colonies of the vector. Visual diagnosis of CTV infection in Key lime seedlings was confirmed by serological analysis of the inoculated material. Under field conditions the pattern of natural spread of CTV is seasonal, with winter, spring, late summer, and early fall being the periods of highest infection pressure. The infection of Key lime seedlings used as trap plants in the field was significantly correlated with the period of greatest flight activity of the vector and the number of colonies on the trap plants. The possibility of using mild strains of CTV in virus control strategies is discussed. Citrus tristeza virus has long One approach, which has been been known to be highly unstable initiated in the Argentine-German (2, 8). Since the tristeza calamity project, is aphid transmission in the 1940's in South America and studies from "dec1inamiento"- the replacement of sour orange as affected and apparently healthy a rootstock for sweet orange, field trees, as well as field trans- grapefruit and tangerine, a num- mission tests. One obstacle, which ber of new strains have formed, interfered with these studies, is which attacked formerly resistant that tristeza is endemic in Misi- scion-rootstock combinations. A ones, as it is in all major citrus pro- critical situation for the citrus in- duction areas of South America. dustry in some locations of south- INOCULATION EXPERIMENTS ern Brazil is due to the appearance of severe tristeza strains with new Four groups of virus-free test epidemiological properties (4). plants (Key lime, trifoliate orange, The symptomatology of "de- 'Calder6n' sweet orange on trifoli- clinamiento", which occurs in ate orange rootstock ; and 'Koethen' Misiones, has some features in sweet orange on trifoliate orange common with severe tristeza. rootstock) were artificially inocu- Therefore, we felt it worthwhile lated with tristeza isolates from ob- to investigate the question, wheth- viously declining and apparently er tristeza is linked with "declina- healthy field trees of 'Calder6n7 miento", as has been suspected by sweet orange on trifoliate orange various experts (5, 6, 7). rootstock. Attempts to transmit

-- 'Research carried out at the Argentine-German Citrus Research Project in Monte- carlo, Misiones, Argentina, with the support of the National Institute of Agricultural Technology, INTA (Argentina), the German Agency for Technical Cooperation, GTZ (Fed. Rep. of Germany), and the Plant Protection Institute of the University of Bonn (Fed. Rep. of Germany). Tristexa and Related Diseases 29 the virus isolates were made using efficiency of different instars of T. 30-50 individuals of the tristeza citricida, single individuals or vector, Toxoptera citricida per test groups of 10 or 30 aphids of each plant. The aphids, reared under of the larval, wingless or winged screen on virus-free Key lime stages of insect were used in sepa- plants, were allowed an acquisition rate experiments. Acquisition and period of 24 hours on Key lime inoculation feeding periods were plants previously infected with 24 hours. The virus source plant field isolates. Subsequently, they had been inoculated with a tristeza were transferred to the test plants, isolate from a "declinamiento"- where they were allowed an in- affected field tree. The results of oculation access period of 24 hours this study are compiled in table 2. before being killed by insecticide. a) All 3 stages were capable of The transmission success was transmitting tristeza, either as evaluated by reading symptoms single individuals or as colonies. and the enzyme-linked immuno- b) Wingless aphids appear to be sorbent assay (ELISA) (1). slightly more effective in their Table 1 summarizes the results. transmission than the winged a) Almost every test plant group stage. In a separate experiment, became partially or totally in- evidence was obtained that trans- fected from both virus sources. b) ovarial passage of these tristeza Sweet orange plants on trifoliate isolates did not occur. orange rootstock did not develop FIELD TESTS symptoms during the observation period of 2 years, but CTV in- Tristeza transmission from fection of many plants was con- "dec1inamiento"-diseased trees firmed by ELISA. c) Key lime under natural conditions was in- control plants, on which aphids vestigated in a trap plant experi- from the stock culture had fed, did ment. Virus-free, screenhouse- not show symptoms, nor did the raised Key lime seedlings were plants used for rearing aphids. placed in groups of 4 plants at two For testing the transmission locations in each of two declining TABLE 1 TRANSMISSION OF CITRUS TRISTEZA VIRUS FROM HEALTHY APPEAR- ING (1-9) AND DECLINING (10 + 11) SWEET ORANGE TREES ON TRIFOLI- ATE ORANGE ROOTSTOCK TO 'KEY LIME', 'CALDERON' SWEET ORANGE AND 'KOETHEN' SWEET ORANGE TRIFOLIATE ORANGE TEST PLANTS BY TOXOPTERA CZTRZCZDA

'Key lime' 'Cald.'/P. trif. 'Koethen'lP. trif. Source Leaf Leaf Leaf Expt. no. plant sympt. ELISA sympt. ELISA sympt. ELISA

1 healthy 3/4* 414 014 214 014 114 2 healthy 414 014 314 014 214 3 healthy 414314 214 014 014 014 214 4 healthy 414 414 014 314 014 414 5 healthy 414 214 014 314 014 214 6 healthy 314 314 014 414 014 214 7 healthy 314 414 014 314 014 314 8 healthy 314 414 014 114 014 214 9 healthy 014 114 014 214 014 214 10 declining 414 414 014 014 014 114 11 declining 414 414 014 - 014 014

* number positivelnumber tested. 30 Ninth IOCV Conference

TABLE 2 TRANSMISSION OF CITRUS TRISTEZA VIRUS BY DIFFERENT MORPHOLOGI- CAL STAGES OF TOXOPTERA CITRICIDA FROM INFECTED TEST PLANTS TO HEALTHY KEY LIME

No. of individuals/ Infection Infection Morphological stage plant rate (%)

Larvae

Wingless adults

Winged adults

* number infectedlnumber exposed. orange groves. The 4 plants were moved were replaced immediately kept on supports at an altitude of by new ones. about 1.30 m at pot rim level (fig. Aphid flight activity was as- 1). The exposure time was 3 to 5 sessed with a modified version of weeks and the plants were main- the Moericke yellow water trap tained thereafter in a growth (3). A refilling device prevented chamber at about 23°C for symp- the traps from drying (fig. 2). One tom expression. Test plants re-

Fig. 1. Support system used for field Fig. 2. Refilling device used for yellow exposure of Key lime trap plants. pan aphid traps. Tristeza and Related Diseases 31 water trap was placed in the noted during summer 1980 in neighbourhood of each of the trap Eldorado. plant groups on supports at about Out of 528 trap plants exposed the same height as the trap plants. to natural infection at both experi- The trap plant experiment was mental sites, 133 showed vein clear- conducted for 33 months between ing symptoms. Of the infected March 1978 and December 1980. plants, 28 (19%) developed stem Aphid trapping started in Janu- pitting of which 14 also showed ary 1979, and colony counting in vein corking. Reduced leaf size, September 1979. cupping and leathery condition of Fig. 3 presents the records ob- the leaves, gum impregnation in tained in the field tests on both stem pits and marked stunting of experimental sites. a) From the the plants were constantly associ- data it is evident that there is no ated with the other severe symp- interruption of aphid colonization toms. in the field, and winged aphids can Severe symptoms appeared be captured at any time of the year. twice during the observation b) Aphid-borne tristeza infection period, in winter 1978 and again in can occur throughout the year, al- fall 1979. It is believed, that their though the intensity varies season- occurrence depends on seasons ally. c) In the trap plant experi- with low average temperatures. ment, two main peaks of infection Infection frequency of the trap were recorded, one at the end of plants was significantly correlated winter and in spring, the other in with the flight activity of T. citri- late summer and fall. However, ex- cida (r = 0.64 at Eldorado, r = ceptionally high infection rates out- 0.60 at Mado) and with aphid side these periods can occur, as colonization of the trap plants (r =

N Of inf. 10 colony countlng wale, traps trap tn~lalled slarted plant5 8

N ol inl. n 1rZ.P plants 8

I978 m nr n xn 1979 m m I xu 19RO m n I X . . ,:e: ,:s: .:s : n. 01 aphid cdonios in, of trapped aphis n, 01 infected trap plants Fig. 3. Infection of Key lime trap plants with citrus tristeza virus, aphids captured in yellow water traps and colonizati~nof trap plants by Toxoptera citricida in Eldorado and Mado, Misiones. 32 Ninth ZOCV Conference

0.72 at Eldorado, r = 0.70 at ship. Tristeza as a semi-persistent Mado) at both experimental sites. virus needs the aphids to be in a "settlement mood" for transmission DISCUSSION success. Characteristic differences be- The classification of 'mild' and tween tristeza isolates originated 'severe' virus strains in this study from "dec1inamiento"-aff ected and is only valid with respect to symp- those from healthy rated sweet tom expression on Key lime test orange trees with respect to their plants. Key lime is not useful as a symptomatology on aphid-infected differential host for assessing the Key lime test plants have not been virulence of tristeza strains on stated in our study. Hence, the ex- other Citrus spp. Therefore, the se- periments did not give an answer lection of mild strains for cross to the question, if tristeza is linked protection control strategies can- with the etiology of the "declina- not be based on Key lime reactions. miento"-phenomenon. For this purpose it is inevitable to An important finding in the test their behaviour in serial trans- field trials was, that vector trans- fers on the scion-rootstock com- mission of tristeza can happen at bination itself, that has to be pro- any time of the year, with the ex- tected. ception of extended periods of hot ACKNOWLEDGMENTS and drv weather durin~summer. he" high positive correlation We are greatly indebted to Ing. between trap plant colonization by Agr. L. Marmelicz and Ing. Agr. J. T. citricidus and the tristeza in- Krausemann for their help in ex- fection rate can be explained with posing trap plants and collecting the specific virus-vector relation- aphid samples from orchard trees.

LITERATURE CITED 1. CLARK, M. F., and A. N. ADAMS 1977. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen. Vir. 34: 475-483. 2. GRANT, T. J. 1959.. Tristeza virus strains in relation to different citrus species used as test plants. Phytopathology 49 : 823-827. 3. MOERICKE. V. 1951. ~ineFarbfalle zur Kontrolle des Fluges von BlattlLusen, insbesondere der Pfirsich Blattlaus, Myxodes persicae (Sulz.). Nachrichtenbl. d. Dt. Pflanzenschutzdienstes (Braunschweig) 3 : 23-24. 4. MULLER, G. W. 0. RODRIGUEZ, and A. S. COSTA 1968. A tristeza virus complex severe to sweet orange varieties. p. 64-71. In Proc. 4th Conf. IOCV, Univ. Florida Press, Gainesville. 5. MULLER, G. W., D. S. RODRIGUEZ, M. A. ROSILLO, and A. S. COSTA 1974. 0 "declinamiento" dos citros nas regioes de Misiones e Corrientes, Argen- tina. Mimeogr. 35 pp., Inst. AgronBmico, Campinas, Sao Paulo, Brazil. 6. PUJOL, A. R., R. E. SCHWARZ, M. V. FERNANDEZ VALIELA, and D. S. RODRIGUEZ 1972. A decline of citrus on trifoliate orange rootstock associated with tristeza virus, p. 154-156. In Proc. 5th Conf. IOCV, Univ. Florida Press, Gaines- ville. 7. ROSSETTI, V., 0. RODRIGUEZ, G. W. MuLLER, T. NAMEKATA, J. POMPEU, and 0. F. MELLO 1970. Relat6rio da visita de thcnicos brasileiros B citricultura da regiiio de Misiones, Argentina. Mimeogr. 25 pp., Inst. AgronBmico, Campinas, Siio Paulo, Brazil. 8. SCHWARZ, R. E. 1979. Die Tristeza-Krankheit von Zitrus in vier Erdteilen-Ein Vergleich. Der Tropenlandwirt 80 : 29-37.