Susceptibility of Citrus Varieties, Species, Citrus Relatives, and Non-Rutaceous Plants to Slash-Cut Mechanical Inoculation with Citrus Tristeza Virus (CTV)' G

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Susceptibility of Citrus Varieties, Species, Citrus Relatives, and Non-Rutaceous Plants to Slash-Cut Mechanical Inoculation with Citrus Tristeza Virus (CTV)' G. W. Miiller and S. M. Garnsey

ABSTRACT. The susceptibility of 47 citrus species, cultivars, hybrids and relatives to citrus tristeza virus (CTV) infection was tested by stem-slash inoculation of greenhouse-grown plants with concentrated, partially purified preparations of CTV. In- fection in 26 of 31 citrus selections inoculated was detected by symptoms and/or by ELISA (enzyme-linked immunosorbent assay). The ELISA procedure was valuable for detecting symptomless infections. CTV-induced vein clearing was observed in several normally latent hosts such as Rangpur lime at the onset of systemic infection. High rates of infection were observed in Etrog citron, Rangpur lime, calamondin, Mexican lime, sweet orange and Citrus hystrix. Grapefruit and Cuban Shaddock were difficult to infect. Six of 16 citrus relatives mechanically inoculated were infected: Aegle marmelos (L.) Corr., Aeglopsis chevalieri Swing., Afraegle paniculata (Schum.) Engl., Citropsis gilletiana Swing. & M. Kell., Microcitrus australis (Planch.) Swing., and Pamburus missionis (Wt.) Swing. No CTV infections were detected by ELISA in the 55 different woody and herbaceous nonrutaceous plants inoculated. Tristeza was re- transmitted mechanically from A. marmelos to Etrog citron. Since the 1940's a large number further possibilities to test citrus, of citrus types and a few citrus citrus relatives and nonrutaceous relatives have been tested for sus- plants as hosts of CTV. We hoped ceptibility to the citrus tristeza that herbaceous hosts could be dis- virus (CTV), at the Instituto covered which would be better as- Agron6mic0, Campinas, Brazil (3, say and/or increase plants for 5, 10, 11). Inoculations have been CTV. made by grafting, by aphid vectors, In this paper, we report the re- and with several species of dodder sults of inoculating and indexing (1, 3). Graft transmission of CTV numerous citrus, citrus relatives, has been limited to hosts graft-com- and nonrutaceous plants to deter- patible with citrus (13), but CTV mine their susceptibility to CTV has been transmitted to several infection by mechanical inocula- citrus relatives, such as Aeglopsis tion. chevalieri by aphids. Repeated at- tempts also have been made to ex- MATERIALS AND METHODS tend the host range of CTV out- Plant materials and growing side the Rutaceae (6, 12), but this conditions. Tests were conducted has been successful only with Passi- in an air-cooled, partly shaded flora gracilis Jacq. inoculated by glasshouse in Orlando, Florida. aphids (1, 14). Temperatures ranged from 22 to The recently developed pro- 27 C in winter and spring and 22 cedure to mechanically transmit to 35 C in summer. Plants were CTV to citrus (7) and the adapta- grown in a sterilized potting mix, tion of the ELISA test to quickly fertilized and sprayed as needed to detect CTV infections (2) offered maintain healthy, vigorous growth. Plants were grown from seeds or *Use of a company or product name from rooted cuttings of clonal, by the U.S. Department of Agriculture virus-free sources. does not imply approval or recommenda- tion of the product to the exclusion of A variety of nonrutaceous others which may also be suitable. plants were chosen for testing. Ninth IOCV Conference

Some were woody, some were hosts to 9 ml of inoculum was recovered for other viruses, viroids, and pro- from 25 g of bark tissue. karyotic pathogens (15- 16), some Inoculation. Most inoculations were weeds found in citrus were done from January through orchards, and one has been de- April and during September and scribed as a nonrutaceous host of October 1980, but some were also T. citricida (17). Etrog citron done in the spring of 1981,1982 and plants which are susceptible to 1983. Inoculations were done by the CTV infection by mechanical in- stem-slash method (7) with 30 oculation (7) were included in cuts at each of 3 sites on the plant every experiment to verify in- stem. Most stems were 0.4 to 0.8 fectivity of the inoculum. cm in diam at the point of inocula- Inoculation preparation. Young tion. Usually a minimum of 10 stem bark from greenhouse-grown plants of each type were inocu- Etrog citron or sweet lime plants lated. Fresh, partially purified infected with CTV isolate T-3 (a preparations were used for each in- seedling yellows (SY) isolate) or oculation test and Etrog citron CTV isolate T-4 (a non-SY isolate), plants were used to verify infectivi- was peeled and wrapped in 5 g ty of each batch. bundles with "Parafilm@." The Initially, cuts on citrus and bundles were sectioned on a Hook- citrus relatives were wrapped with er@microtome and finely diced with Stericrepea (Beacon & Janis Ltd., a razor blade at a 1:3 (w/v) ratio London, W.C.I., England). Later, in extraction buffer (0.05 M Tris- however, this was found unneces- HC1 buffer [Tris (hydroxymethyl) sary. Some succulent nonrutaceous aminomethane hydrochloride], pH plants were covered with plastic 7.8 which contained 100 mg/l su- bags and on others the inoculation crose and 2 pl/ml of 2-mercapto- cuts were wrapped with "Parafilm" ethanol). The extract was squeezed and the plants were not covered. through cheesecloth by hand press Leaf midribs were also slashed on and reextracted with a small some herbaceous plants. After in- volume of buffer. All extraction oculation, the herbaceous plants steps were done at 0 to 4 C. Ex- were put in the shade for 48 hours tracts were clarified by centrifuga- before the plastic bags were re- tion for 15 minutes at 12,000 RPM moved. in a Sorvall@ SS-34 rotor and Citrus and most woody plants layered in 2.5 x 7.5 or 2.5 x 8.75 cm were cut back immediately to force centrifuge tubes containing a fresh- new growth and the herbaceous ly prepared sucrose step gradient plants were cut back 2 weeks after (5 ml of 250 mg/ml and 5 ml of 600 inoculation. Rapidly growing plants mg/ml sucrose in Tris buffer) (8). were cut back several times before Tubes were centrifuged overnight testing for CTV infection. Three to at 21,500 RPM in a Beckmans 5 uninoculated control plants were SW25.1 rotor or at 19,000 RPM in kept for each species tested. a SW28 rotor. Half ml fractions Assay for infection. All citrus were collected in premarked test cultivars, species and relatives were tubes by puncturing the bottom of examined periodically for vein the gradient tube. Gradient frac- clearing and the noncitrus plants tions were tested for CTV content were examined for vein clearing by ELISA (bead procedure) and plus any other abnormalities. the leading (lowest) 2-3 CTV-con- After the Etrog citron control taining fractions were combined plants showed vein-clearing symp- and diluted I :l with 0.05 M Tris to toms, succulent stem bark tissue use as inoculum. Approximately 6 was collected from inoculated citrus Tristexa and Related Diseases and citrus relatives. Whole young vein-clearing symptoms not pre- stem was collected from succulent viously reported were observed in nonrutaceous hosts. Chopped tissue Rangpur lime (fig. 1A) 40-50 days (0.5 g) was placed in a 2.5 cm-di- after inoculation. Faint vein clear- ameter glass tube with 5 ml of ing was observed in Rusk citrange PBST-2 PVP buffer (phosphate- and in some sweet orange plants. buffered saline with 0.05% Tween@ The initial vein clearing was more 20 and 2% polyvinylpyrrolidone,, pronounced than that in subsequent m.w. 40,000) (2, 4) and homogen- flushes, especially in plants which ized about 15 seconds with a SDT- are normally asymptomatic. The Tissumizer@ (Tekmar Co., Cin- other citrus cultivars and types cinnati, Ohio 45222). The extracts showed varying rates of infection. were filtered through glass wool Cleopatra mandarin, Duncan grape- and the ELISA tests were con- fruit, Cuban Shaddock, and Gomri ducted by the double antibody sand- and Soh jhalia rough lemon se- wich method (4). The CTV anti- lections were not infected (table serum used was prepared to un- 1). fixed, whole virus of the T-4 iso- Six of 16 citrus relatives tested, late (9). IgG was purified and con- Aeglopsis chevalieri, Aegle mar- jugates to alkaline phosphatase melos, A fraegle paniculata, Citrop- prepared as described previously sis gilletiana, Microcitrus australis (2, 4). We used polystyrene Micro and Pamburus missionis, were in- !ELISA@ plates (Dynatech Labs, fected. Rates of infection were Inc., Alexandria, Virginia 22314) lower than in most citrus selections with round-bottom wells. Healthy (table 1) ; however, the infected extracts, CTV-infected extracts, plants of A. chevalieri, A. marmelos and buffer controls were included and A. paniculata which became in- in each plate for reference. Plates fected showed very clear, persistent were scored visually and results re- vein-clearing symptoms (fig. 1B). corded by photography. In some Tristeza was partially purified cases, the absorbance at 405 nm from A. marmelos and was re- was also determined spectrophoto- transmitted mechanically to Etrog metrically on diluted samples to citron. verify assessment. Samples were Inoculation of nonrutaceous not considered CTV-positive unless plants. Plants of 55 species belong- the O.D.,,, was 2X that of the ing to 26 families outside the Ruta- healthy controls. ceae were inoculated with CTV. RESULTS They are listed alphabetically by family as follows : AMARANTHA- Inoculation of citrus varieties, CEAE-Gomphrena globosa L.; species and relatives: Over 600 ANACARDIACEAE - Brazilian plants comprising 47 citrus varie- pepper tree (Schinus terebinthio- ties, species and relatives were me- folius Raddi) ; APOCYNACEAE- chanically inoculated with CTV Cantharanthus roseus L.; ARA- (table 1). Calamondin, Mexican CEAE-DiefSenbachia sp. ; BUXA- lime, Rangpur lime and Etrog CEAE-Buxus sempevvirens L. cv. citron showed the highest incidence Wintergreen ; CAPRIFOLIA- of CTV infection. Some Mexican CEAE-Viburnum dentatum L., lime plants showed CTV vein clear- and Viburnum odoratissimum Ker ; ing within 30 days after inocula- CHENOPADIACEAE - Cheno- tion, and Etrog citron plants often podium amaranticolor Costa & showed vein-clearing symptoms 35 Reyn, Chenopodium quinoa Wild, to 45 days after inoculation (in the and Beta vulgaris L. cvs. Early second flush of growth). Clear-cut, Wonder and Swiss Chard ; COLEA- 36 Ninth IOCV Conference

Fig. 1. Citrus tristeza virus-induced vein clearing in mechanically inoculated Rangpur lime (A) and Aegle marmelos (B).

CEAE - Ligustrum japonicum L. and Hibiscus esculentus L. cv. Thunb. and Ligustrum sinense Clemson spineless ; MORACEAE Lour.; COMPOSITAE - Bidens Osage orange (Maclura pomifera pilosa L., Chrysanthemum mori- (Raf.) Schneid) ; MYRSINA- folium Ramat, Gynura aurantiaca CEAE - Ardisia crispa (Lam.) DC, Gynura sarmentosa DC, and A. DC; PASSIFLORACEAE - Zinnia elegans Jacq. cv. Pepper- Passiflorn gracilis Jacq., and Passi- mint, Wedelia trilobata Hitchc.; flora sp. ; PEDALIACEAE-White CUCURBITACEAE - Momordica sesame (Sesamum indicum L.) ; balsaminn L. ; ELAEGNACEAE- PITTOSPORACEAE - Pitto- Eleagnus commutata Bernh. ; ERI- sporum tobira Ait tobira cv. varie- CACEAE - Rhododendron indi- gated ; PODOCARPACEAE - cum (L.) cv. Formosa, and R. ob- Podocarpus sp.; ROSACEAE - tusum (Lindl.) cv. Christmas Malzls sylvestris Mill cv. York; Cheer ; EUPHORBIACEAE - RUBIACEAE - Ixora spp., Acalypha wilkesiana Muell. Arg.; Pentas spp., and Gardenia veitchii LABIATEAE - Coleus blumei Hort. ; SOLANACEAE - Nicoti- Benth.; LEGUMINOSE - Phase- ana clevelandii Gray, Nicotiana olus vulgaris L., Crotalaria spect- edzvardsonii Christie & C. W. Hall, abilis Roth and Cassia sp.; MAL- Nicotiana glauca Graham, Nicoti- VACEAE - Hibiscus rosa sinensis ana glutinosa L., Nicotiana megalo- Tristeza and Related Diseases 37

TABLE 1 TRANSMISSION OF CITRUS TRISTEZA VIRUS (CTV) BY MECHANICAL INOCULATION TO CITRUS CULTIVARS AND RELATIVES

No. plants % No. plants yo Citrus variety inoculated infection Citrus relative inoculated infection

Calamondin 10 100 Citropsis gilletiana 10 Mexican lime 15 87 Afraegle paniculata 35 Etrog citron (S-1) 40 80 Microcitrus australis 20 Rangpur lime 20 75 Aegle marmelos 26 Palestine sweet lime 10 70 Aeglopsis chevalieri 36 C. webberi 10 70 Pamburus missionis 20 Etrog citron (RMA 861) 36 64 Ataluntia monophylla 10 Iran lemon 10 60 Balsamocitrz~s dawei 5 C. hystrix 10 60 Clausena ezcavata 5 Valencia orange 10 50 Clausena lansium 1 Gou Tou sour orange 10 50 Glycomis pentaphylla 10 Florida rough lemon 10 50 Hesperethusa crenulata 5 Rusk citrange 10 50 Mz~rrayakoenigii 14 Alemow 10 50 Murraya paniculata 10 C. karna 9 44 Swinglea glutinosa 10 Navel orange 17 35 Triphasia trif oliata 10 Coorg lime 10 30 Sour orange 40 30 C. excelsa 10 30 Temple tangor 10 30 Milam lemon 15 27 C. myrtif olia 10 20 Soh Jhalia (rough lemon) 10 10 Eureka lemon 20 10 Volkamer lemon 10 10 Orlando tangelo 10 10 Marsh grapefruit 21 5 Cleopatra mandarin 10 0 Duncan grapefruit 20 0 Gomri rough lemon 10 0 Cuban Shaddock 10 0 siphon Heurck & Muel, Nicotiana DISCUSSION tabacum L., Solanum nigrum L., Lycopersicon esculentum Mill. cv. Although high rates of in- Patio, Capsicum annuum L. cv. fection are sometimes obtained, the California Wonder, Datura metel mechanical inoculation procedures L., Daturu stramonium L., and used in these studies are not high- Solanum seaforthianum Andr. ; ly efficient for transmitting CTV SCROPHULARIACEAE - Scopo- to most citrus hosts. Many different liu sinensis Hensl., and Antirrhi- species of citrus can become in- num majus L. fected with CTV by extensive me- No symptoms which would in- chanical inoculation with concen- dicate CTV infection were ob- trated in vitro sources of inoculum ; served, except for some vein clear- however, accidental transmission ing in H. rosa sinensis, which was of CTV as a contaminant on cutting also present in noninoculated tools in the field is less likely with controls. All nonrutaceous plants unconcentrated inoculum and few- (many of which were assayed er wounds. twice) tested negative for CTV in- Partially purified tristeza iso- fection by ELISA. lates can be inoculated directly to Ninth IOCV Conference plants of interest without need for diagnosis of CTV infection in these an easily infected, intermediate citrus relatives made previously by host such as Etrog citron. This may symptoms, and also confirm the be of special interest for introduc- utility of ELISA for indexing for ing mild CTV isolates from one CTV infection in plants which are country to another in purified form not graft-compatible with citrus for cross-protection studies or indicators. other experimental uses. In most cases, we did not at- The mechanical transmission of tempt to infect the citrus relatives CTV between citrus and citrus rela- which were nonsusceptible to me- tives which are not graft com- chanical inoculation by other patible will also permit use of these means. However, we did find that alternate hosts for virus increase CTV could not be detected in graft- purposes should they prove es- inoculated Swinglea glutinosa pecially suitable host plants. Be- plants by ELISA. cause mechanical inoculations to Although we had only negative normally receptive hosts, such as results with nonrutaceous plants, Etrog citron, often give somewhat further tests may yet show that erratic results, the relative rates some of these are hosts of CTV. of infection reported are signifi- Additional genera of the Ruta- cant only in a general sense. How- ceae, such as the genus Fagara ever, it appears that some citrus which has a number of species in cultivars such as grapefruit are the Amazon area of Brazil, could be more difficult to infect than others tested. Plants in other families re- such as Etrog citron or Mexican lated to the Rutaceae are other lime. Negative results for indi- possible candidates. vidual selections where only a The stem-slash inoculation pro- single test was made should not be cedure may not be highly suitable regarded as definitive at this point; for some of the nonrutaceous however, in all cases reported, the plants tested. We did not infect same inoculum caused extensive in- Passijlora gracilis even though fection in known susceptible hosts. more than 50 plants were inocu- No striking differences were noted lated in several tests. Since it is a between the 2 isolates tested. Vari- known host of CTV which is infect- ations between preparations of the able by aphids and the inoculum same isolate were as great as vari- used here was infectious, it appears ations between isolates. Accurate that the slash-cut procedure was estimates of the concentration of not appropriate for this host. Other intact, infectious particles are sap inoculation procedures, includ- difficult to obtain and generally ing leaf-abrasion inoculation have were not made on the inocula used either not been successful or were here. less successful than the stem-slash A. chevalieri, A. paniculata and method for inoculating citrus P. missionis have all been previous- (Garnsey and Miiller, unpublish- ly reported as reactive hosts of ed), and we did not have sufficient CTV (12, 13), and CTV has been inoculum to test numerous different transmitted from the former 2 procedures on different hosts. species to citrus by aphids (12). The use of ELISA was a great Aegle marmelos was reported non- advantage for indexing for CTV reactive to aphid inoculation with infection in symptomless plants, CTV by Knorr (12), but was in- especially nonrutaceous ones. We fected in our tests. Positive sero- believe that if any of the nonruta- logical reactions in ELISA with ceous plants tested had been highly CTV specific antibodies confirm the susceptible to CTV infection and Tm'stexa and Related Diseases 39 able to support CTV multiplication, Whidden. Also acknowledged are we should have been able to detect Dr. Robert Knight of the Miami this. Although ELISA may not de- Plant Introduction Station; Dr. M. tect very low levels of infection, Cohen, University of Florida, Fort we detected CTV in citrus extracts Pierce, Florida; and Dr. J. B. at dilutions 100 to 200X those used Carpenter, USDA, Indio, Cali- for the assays here. fornia, for furnishing plant ma- terial; and Dr. Condorcet Aranha, ACKNOWLEDGMENTS Department of Economic Botany, The authors gratefully ac- Instituto Agron8mic0, Campinas, knowledge the expert technical as- Brazil, for assistance in classifying sistance of C. Henderson and R. the nonrutaceous plants.

LITERATURE CITED 1. BAR-JOSEPH, M., S. M. GARNSEY, and D. GONSALVES 1979. The closteroviruses: A distinct group of elongated plant viruses. Adv. Virus Res. 25: 93-168. 2. BAR-JOSEPH, M., S. M. GARNSEY, D. GONSALVES, and D. E. PURCIFULL 1980. Detection of citrus tristeza virus. I. Enzyme-linked immunosorbent assay (ELISA) and SDS-immunodiffusion methods, p. 1-8. In Proc. 8th Conf. IOCV, Riverside, California. 3. BENNETT, C. W., and A. S. COSTA 1949. Tristeza diseases of citrus. J. Agric. Res. 78: 207-237. 4. CLARK, M. F., and A. N. ADAMS 1977. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant virus. J. Gen. Virol. 34: 475-483. 5. COSTA, A. S., T. J. GRANT, and S. MOREIRA 1949. InvestigacCies sobre a tristeza dos citros. 2. Conceitos e dados sobre a reacao das plantas citricas B tristeza. Bragantia 9: 59-80. 6. COSTA, A. S., G. W. MULLER, and C. L. COSTA 1968. Rearing the tristeza vector Toxoptera citricida on squash, p. 32-35. In Proc. 4th Conf. IOCV, Univ. of Florida Press, Gainesville. 7. GARNSEY, S. M., D. GONSALVES, and D. E. PURCIFULL 1977. Mechanical transmission of citrus tristeza virus. Phytopathology 67 : 965-968. 8. GARNSEY, S. M., R. F. LEE, and R. H. BRLANSKY 1981. Preparation and stability of infectious citrus tristeza virus (CTV). Phyto- pathology 71 : 218. (Abstr.) . 9. GONSALVES, D., D. E. PURCIFULL, and S. M. GARNSEY 1978. Purification and serology of citrus tristeza virus. Phytopathology 68: 553-559. 10. GRANT, T. J., and A. S. COSTA 1948. A progress report on studies of tristeza diseases of citrus in Brazil. I. Behavior of a number of citrus varieties as stocks for sweet orange and grapefruit, and as scions over sour orange rootstock when inoculated with tristeza virus. Proc. Fla. State Hort. Soc. 61: 20-33. 11. GRANT, T. J., A. S. COSTA, and S. MOREIRA 1949. Studies of tristeza disease of citrus in Brazil. 111. Further results on the behavior of citrus varieties as rootstocks, scions and seedlings when inoculated with the tristeza virus. Proc. Fla. State Hort. Soc. 62: 72-79. 12. KNORR, L. C. 1956. Suscepts, indicators, and filters of tristeza virus, and some differences between tristeza in Argentina and in Florida. Phytopathology 46 : 557-560. 13. McCLEAN, A. P. D. 1961. Transmission of tristeza virus to Aeglopsis chevalieri and Afraegle panisulata. S. Afric. J. Sci. 4 : 83-94. 14. MULLER, G. W., A. S. COSTA, E. W. KITAJIMA, and I. J. B. CAMARGO 1974. Additional evidence that tristeza virus multiplies in Passiflora spp., p. 75-78. In Proc. 6th Conf. IOCV, Univ. California, Div. Agr. Sci., Rich- mond. 15. PLESE, N., and D. MILICIC 1973. Two virus isolates from Maclura pornifera. Phytopathol. Z. 77: 178-183. 40 Ninth ZOCV Conference

16. WEATHERS, L. G., S. M. GARNSEY, A. CATARA, P. R. DESJARDINS, G. MAJORANA. and H. TANAKA 1974. ~ec'hanicaltransmission of citrus viruses, p. 147-156. In Proc. 6th Conf. IOCV, Univ. California, Div. Agr. Sci., Richmond. 17. YUKI, V. A., G. W. MULLER, J. A. BETTI, and A. S. COSTA 1978. Maclura aurantiaca hospedeira nPo rutbceae do afideo vetor Toxoptera citn'cida. Resumos do 111 Congress0 Latino American0 de Entomologia, IlhBus/Itabuna, BA.