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Lack of Cross-Protection Between Citrus Exocortis Vir- oid and Citrus Viroids Associated with Mild Symptoms in Etrog Citron* S. M. Garnsey, R. F. Lee, and J. S. Semancik ABSTRACT. Etrog citron plants systemically infected with citrus viroid (CV) isolates, which cause mild symptoms in citron, developed the same typical severe symptoms of citrus exocortis viroid (CEV) infection as noninfected plants when challenge-inoculatedby graft or stem-slash inoculation with CEV. There was no difference inincubation period for expression of symptoms ofthe challenge isolate between plants with a primary infection of a mild viroid and noninfected plants. When extracts from citrons infected with the viroids inducing mild symptoms were subjected to sequential gel electrophoresis (sPAGE) under native and denaturing conditions and silver stained, no CEV band was found, but bands corresponding to citrus viroids CV-IIa and CV-IIIb were present. No hybridization was observed between cDNAprobes to CEV and either mild citronviroid. The lackof cross-protection, or interference by mild viroids with infection by the CEV challenge, supports the lack of relationship between CEV and the other mild citrus viroids studied. Index words. sequential gel electrophoresis, stem-slash inoculation Etrog citron was used extensively tristeza virus (13), and cross-protec- for testing citrus for exocortis infection tion had been reported between mild following its introduction as a diagnos- and severe strains of potato spindle tic indicator for citrus exocortis (4). tuber viroid (PSTV) (2,14) and be- Some variation in symptom expression tween citrus exocortis viroid (CEV), in citron indicators was noted (17), and PSTV, and chrysanthemum stunt some inoculum sources caused only (CSV) viroids (14) in tomato or very mild symptoms (8,18). At the chrysanthemum. Infectivity assays time, it was assumed that the causal and purification attempts by PAGE in- agent of exocortis was a virus and that dicated that the mild isolates were the variation in symptom expression present in lower concentrations than indicated presence of isolates or strains CEV in comparable tissue (1,8). It was of different severities. The viroid na- also observed that mild symptoms ture of exocortis later became evident, could be obtained by infecting less reac- but the assumption remained that the tive clones of citron with CEV (8). mild symptoms caused by some isolates These observations suggested that reflected strain differences among a failure of cross-protection could be as- single viroid pathogen. sociated with either poor distribution Several cross-protection experi- or poor replication of the protectingiso- ments were initiated in Florida to test late, and this was investigated in sub- different isolates which produced mild sequent experiments. symptoms for ability to protect cit- As these protection studies were rons against several isolates that pro- progressing, evidence accumulated duced classical severe symptoms. No that the symptoms in Etrog citron, evidence was obtained for cross-pro- which were once all attributed to CEV, tection in these tests. These results were caused by several viroids with were puzzling since cross-protection distinct differences in molecular had been reported in citrus with citrus weight and other properties (3,6). Examination of the viroid isolates used in the various cross-protection studies *Mention of a trademark, warranty, prop- by sequential PAGE (sPAGE) proce- rietary product, or vendor does not constitute a dures revealed that there were several guarantee by the U.S. Department of Agricul- ture and does not imply its approval to the exclu- viroid species present, and that the sion of other products or vendors that may also mild isolates test for protection did not be suitable. contain typical CEV. Twelfth IOCV Conference Recent reports have been pub- Agriculture and Consumer Services, lished on interference or protection be- Division of Plant Industry, code Te 20- tween two CEV isolates (7) and be- 3-5) to Etrog citron and subsequent tween members of the CV-I1 group passage by slash-cut transmission to (15,22). other Etrog citrons. It also causes typ- This paper reports that cross-pro- ical, severe CEV symptoms in citron tection did not occur between CV-I1 or and contains CEV, CV-IIa, and CV- CV-I11 and CEV. The lack of cross- IIIb (23). Source El0 was obtained protection observed is a further indica- originally by graft-inoculation to Etrog tion of a lack of relationship between citron from a Persian lime tree on Group I1 and Group I11 citrus viroids Rangpur lime rootstock and sub- which cause mild symptoms in Etrog sequent passage by slash-cut inocula- citron and CEV. A preliminary report tion to Etrog citron. It produces only ofthese results has been presented (9). mild symptoms in Etrog citron. Ex- tracts from ElO-infected plants do not MATERIALS AND METHODS carry CEV, but contain CV-111. Source Ell was obtained by graft-inoculation Host plants. All cross-protection to citron from a propagation of the Tem- studies were done with clonal propaga- ple orange Te 20-3-5 (see E22), which tions of three selections of Etrog citron: had undergone heat therapy treatment. Arizona 861,861-S1, and OES-4. Their It was also transferred by mechanical responses to CEV infection have been inoculation to citron. Ell causes mild described (8,18). All receptor plants symptoms in Etrog citron similar to were propagated as rooted cuttings those produced by E10. Extracts from and grown in individual containers in Ell-infected plants containboth CV-I1 a sterilized potting medium. Plants and CV-111. The CV-I1 is apparently were maintained in a partially shaded CV-IIa and not CV-IIb (21) since the glasshouse cooled with an evaporative original source tree had been propa- cooling system. Temperature condi- gated on Orlando tangelo for more than tions during the periods of experimen- 10 yr without cachexia symptoms (16). tation followed a normal diurnal fluctu- Isolates E16b and E25 used in one li- ation with night temperatures of 21-24 mited test produce typical CEV C and day temperatures of 27 to 34 C. symptoms in Etrog citron, but pres- Plants were fertilized as required to ence of other viroids was not deter- promote vigorous growth and sprayed mined. periodically to control pests. Precau- Inoculation procedures. Graft-in- tions were observed in handling and oculation was done by conventional T- pruning plants to avoid accidental vir- bud procedures with a chip of stem tis- oid infection by contamination (10,17). sue as the inoculum source. Mechanical Cutting tools were dipped in diluted inoculation was done by a stem-slash sodium hypochlorite or a mixture of procedure (10). The cutting blade was formalin and sodium hydroxide (10). contaminated either by dipping it into Viroid isolates. Four viroid an extract from infected plants, or by sources were used extensively in vari- cutting into the stem of a viroid-in- ous tests. Isolate E9 is a standard iso- fected plant, and then immediately late of CEV, which has been used in making a cut in the stem of the recep- many studies, and is also entered in the tor. Ten cuts were made per plant with collection of the American Type Cul- the blade freshly contaminated prior ture Collection as PV194. In our as- to each cut. Receptor plants were cut says, and in other independent tests back following inoculation to force new (12), it has been shown to contain only growth which was confined to a single CEV. It produces typical CEV symp- shoot. toms in Etrog citron. Source E22 was Challenge inoculations to previ- obtained by graft-inoculation from a ously inoculated plants were not made Temple orange tree (Florida Dept. of until definite symptoms of the primary Citms Viroids isolate had been observed, normally 4 within 20 days postinoculation if ex- to 8 months postinoculation. pressed in the first flush. Symptom evaluation. The leaf PAGE and cDNA hybridization epinasty symptoms typical of CEV in- procedures. Bark and leaf midrib tis- fection in citron (Fig. lb) were rated sue from young, symptomatic flushes visually on a 0 to 4 scale where 1 indi- of Etrog citron were powdered inliquid cated a deflection in the leaf blade from nitrogen and extracted immediately or zero to 90 degrees; 2, a deflection of 90 lyophilized for extraction at a later to 180 degrees; 3, a deflection of 180 to date. Extraction, concentration, and 270 degrees; and 4 indicated that the leaf electrophoresis were as described pre- had curled into at least a full circle (360 viously (5,20) and included phenol ex- degrees). A composite score per plant traction, partitioning with 2M lithium was compiled by totaling the reading chloride, partial purification on CF11 for all leaves on a flush and dividing by cellulose, and sPAGE. the number of leaves. Stunting was de- A cDNA probe to a California iso- termined by measuring shoot growth late of CEV was synthesized from the at different intervals after inoculation. viroid template by random priming in Incubation period was determined as the presence of 32P. Hybridizations the time between challenge inoculation were done as previously described (5). and appearance of symptoms of the RESULTS challenge isolate. Because of the periodicity in growth flushes in citrus, Cross-protection. In the first cross- incubation period data is less precise protection test attempted, Arizona 861 than in rapidly and continuously grow- citron plants systemically infected ing herbaceous plants. Under our con- with El0 and Ell isolates and non- ditions, symptoms of severe isolates of protected controls were challenged CEV normally appeared at the onset with E9. There were nine replications of the second flush of growth following per treatment. In addition, graft and inoculation that was approximately 40 mechanical challenge were tested for to 50 days. Symptoms can appear each combination.

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