The Citrus Exocortis Disease: a Complex of Viroid-Rnas

The Citrus Exocortis Disease: A Complex of Viroid-RNAs

N. Duran-Vila, J. A. Pina, J. F. Ballester, J. Juarez, C. N. Roistacher, R. Rivera-Bustamante, and J. S. Semancik

ABSTRACT. Citrons inoculated with different field sources, displayed a variety of symptoms ranging from very mild leaf bending and necrosis to the severe reaction normally associated with exocortis disease. Nucleic acid preparations from shoot samples were analyzed by sequential polyacrylamide gel electrophoresis. All source from both California and Spain contained one to four viroids with distinct physical and biological properties. The size range was estimated from 371 nucleotides for the citrus exocortis viroid (CEV) to 275 for the smallest viroid. The recovery of single viroids suggested a relationship between the distinct viroids and the symptom reaction expressed in citron. Index words. Citron variable viroid, citrus viroid complex, electrophoresis, nucleic acids, hybridization assays.

Since the first description of the The transmission of severe forms exocortis disease by Fawcett and of the disease to Gynura aurantiaca Klotz (5), degrees of stunting, differ- D.C. and other herbaceous hosts (30), ences in mode of formation of bark permitted the isolation and charac- scaling and time of appearance of terization of the causal agent as a low symptoms on sensitive rootstocks molecular weight, infectious RNA, such as trifoliate orange have been re- the citrus exocortis viroid (CEV) (21, ported. These were attributed to dif- 22). Citrons inoculated with purified ferent strains of the disease agent (2, CEV preparations produced severe 3, 7, 8 , 9, 14, 17). With the transmis- epinasty, leaf rugosity and stunting. sion of the disease to citron, and the Further biochemical and biophysical selection of sensitive citron clones characterization of the causal agent of such as USDCS 60-13, Arizona 861 the exocortis disease (6, 20, 23, 24, and Arizona 861-S1 (1, 16) as indi- 29) has focused on viroid RNA from cators, more sensitivity and faster G. aurantiaca D.C., tomato and exocortis indexing was achieved. In Chrysanthemum morifolium Ram. the process, the use of trifoliate which included, in addition to the 371 orange as the indexing host for the nucleotide viroid (29) some variants exocortis disease was virtually aban- with minor differences in the number doned. Therefore, in most cases the of nucleotides residues ranging from bark shelling reaction in trifoliate 370 to 375 (27, 28). orange has not been reproduced with Isolates producing mild and mod- exocortis disease isolates identified erate reactions on citron did not cause by the citron reaction. symptoms in Gynura. These early ob- Browning on the tips of the leaf servations suggested either the exis- blades, petiole wrinkle and necrosis, tence of many forms of "exocortis dis- midvein necrosis, leaf epinasty and ease", undetectable levels of the stunting are among the symptoms ob- agent, or Gynura as a selective host. served on inoculated Arizona 861-S1 Recently, the "citron variable vir- citrons. All these symptoms were oid (CVaV)" isolate (18). now known considered as diagnostic evidence of to comprise a complex of four viroids mild, moderate or severe strains of with distinct electrophoretic exocortis when detected during in- mobilities have been described along dexing of symptomless stocWscion with other viroid species and shown combinations. to be associated with citron plants dis- Diseases Induced by Viroids and Viroidlike Pathogens playing a variety of symptoms rang- (Arizona 861-S1) with various field ing from mild to moderate in citron sources. The stable citron isolates (4). Since the symptom expression of constitute either single viroids or the CVaV isolate has not been as- mixtures of 2-4 viroids. In many cases sociated with any one or more of the the original field sources are no longer four viroid components, the name available. "citron variable viroid" presents a Polyacrylamide gel electro- misleading terminology. phoresis. Nucleic acid preparations This paper presents additional in- were obtained from shoots of citrons formation describing different viroid inoculated with different field sources RNAs associated with the broad by phenol extraction and lithium "exocortis disease" reactions on cit- chloride partitioning (4, 24). When ron, and a provisional grouping of analyzed by sequential gel elec- these citrus viroids (CV) is proposed trophoresis under native and denatur- based on their physical and biological ing conditions (4, 20) followed by properties. silver staining (lo), one or more viroid RNAs were detected in each sample VIROID PROFILES OF (4). In a previous report (4), an at- CITRON ISOLATES tempt to classify the different field isolates on the basis of their viroid Source materials. The citrus vir- profiles was presented. However, a oids described here were derived new technique, which enhances the from inoculation of Etrog citron resolution of circular viroid molecules

Fig. 1. Polyacrylamide gel electrophoresis of nucleic acid extracts under low pH, denaturing conditions (8M urea) following electrophoresis in 5% gels. Samples were from isolates E144, E152, El51 and El24 from Spain (lanes 1, 3, 5 and 7), and isolates E804, a mixture of E818 and E805 and purified CV-IIb from California (lanes, 2,4 and 6). Small arrows indicate the migration region of CV-111s. Gels were silver stained. 154 Tenth ZOCV Conference

(13), revealed an almost continuous and necrosis, midvein necrosis, leaf array of viroid RNAs with different curling and epinasty, and different electrophoretic mobilities in the area degrees of stunting. Analysis by se- of CV-I1 and CV-I11 (fig. 1) whereas quential gel electrophoresis revealed only these two distinct bands had characteristic viroid profiles as- been recognized earlier. sociated with individual isolates (table The relative mobilities of the vir- 2). oids found in selected sources from Spanish sources in general, con- California and Spain were compared tain more viroids than sources from by co-electrophoresis and a consensus California. This is probably a result catalogue of citrus viroids was estab- of topworking, a common practice in lished. Table 1 describes five major Spain. Top worked trees would ac- groups of citrus viroids defined by cumulate all viroids from the different similar electrophoretic mobilities. budwood sources into a single tree. This was estimated by comparison of In contrast, single viroids were found their relative migration in denaturing in sources from California where top- PAGE with the citrus exocortis viroid working is not a common practice. (CEV) and the avocado sunblotch vir- Some viroids such as CEV, CV- oid (ASV). As shown later, the com- IIa, and CV-IIIb are widespread in ponents of each group also display both California and Spain, whereas similar physical and biological proper- CV-Ib, CV-IIIa and CV-IV are re- ties. stricted to a few sources from Califor- Viroid profiles of "exocortis" nia. Another viroid, CV-IIb, found isolates found in California and only as a component of E821, is the Spain. A series of stable "exocortis" causal agent of the cachexia disese, isolates (judged by symptoms on in- and the main viroid component of the oculated citron plants) were reco- cachexia isolates (25). Two other vir- vered from different citrus sources. oids, CV-IIIc and CV-IIId, were These isolates produced a variety of widespread in the Spanish collection, reactions in citron ranging from very but were not found in California mild leaf necrosis, to petiole wrinkle sources.

TABLE 1 PHYSICAL PROPERTIES OF CITRUS VIROIDS '(CV)

CF-11 CelluloseY cDNAx hybridization Citrus Ethanol Elution viroids Basesz 25% 20% CEV CV-Ib ASBV

CEV CV-Ia cv-Ib CV-IIa CV-IIb (CCaV) CV-IIIa cv-IIIb cv-IIIc CV-IIId

"By comparison with Citrus Exocortis Viroid (CEV) and Avocado Sunblotch Viroid (ASV) in denaturing PAGE. YCF-11 ellulose elution indicates ethanol % to begin specific viroid elution. "Relative reaction with cDNA as determined by slot-blot and electroblot hybridization. "NT = not tested. TABLE 2 DISTRIBUTION OF CITRUS VIROIDS IN STABLE ISOLATES FROM CALIFORNIA AND SPAIN

Viroid content Citron DuplicateY Isolate Reactionz CEV CV-Ia CV-Ib CV-IIa CV-IIb CV-IIIa CV-IIIb CV-IIIc CV-IIId CV-IV Isolates

California: E 811 Var. E 820 5 E 814 5 E 812 5 E 803 2 E 821 3 E 818 1 E 822 3 E 805 2 E 804 4 Spain: E 161 4 E 117 4 E 124 4 E 104 4 E 129 3-4 E 144 2-3 E 151 2 E 107 1-2 E 152 1 E 111 1

"Indexing was performed on Arizona 861-S1 as described in Agricultural Handbook 333, (1). The citron reaction was rated on a 0-5 scale for California isolates and a 0-4 scale for Spanish isolates with 0 = healthy and 4 or 5 = severe. YIsolateswith same profile as isolate listed on left. Tenth ZOCV Conference

PHYSICAL PROPERTIES arations from isolates containing CEV (fig. 2). A weak reaction was Cellulose affinity. The present also detected with preparations from knowledge about the differential source E804 which did not contain binding properties of viroids to eel- CEV, but contained CV-IIIa and CV- lulose (19) indicates that different vir- IV. Hybridization of an electroblot oids begin elution at distinct ethanol from an E804 extract on denaturing concentrations when performing eel- PAGE gave a weak positive reaction lulose chromatography. The elution with CV-IV and no reation with CV- properties of citrus viroids in CF-11 IIIa. cellulose chromatography are sum- A positive reaction was also ob- marized in table 1. These properties served when samples containing CV- can be utilized as a discriminating tool Ia or CV-Ib were hybridized with for the recovery of preparations en- cDNA to CV-Ib. An electroblot pro- riched for specific viroids, or as an ad- cedure was also utilized to confirm the ditional parameter for viroid charac- homology of CV-Ia and CV-Ib. In this terization. case, however, the reaction was very When nucleic acid-cellulose prepa- strong, indicating a high degree of rations containing CV-IIa (RNA-11) homology of CV-Ib for CV-Ia. It is were extensively washed with buffer interesting to note that CV-Ib is re- containing 25% ethanol, as previously stricted to California isolates whereas reported (4)) this viroid was not reco- CV-Ia occurs in both California and vered from the final eluant. However, Spain. Thus, perhaps the smaller CV- we have been able to obtain CV-IIa- Ib may comprise a recent deletion de- enriched preparations by washing the rivation of CV-Ia in California. No cellulose columns first with buffer hybridization was detected with any containing 30% ethanol and eluting of the sam~lestested to cDNA to with 25% ethanol buffer and--or buf- ASV. fer alone. Analysis of the enriched Although these hybridization re- preparations provided evidence for sults do not provide a complete pic- the viroid nature of RNA-I1 (Duran- ture of the sequence homology among Vila et al., unpublished). In addition, citrus viroids, they support the segre- the utilization of CF-11 cellulose gation of citrus viroids into five sepa- chromatography permitted the dis- rate groups. Additional hybridization crimination of CV-IIIa and CV-IIIb tests using probes made to a CV-I11 even though these have very close viroid, and, ultimately, base sequenc- electrophoretic mobilities. ing is needed to define sequence re- Sequence homology based on lationships among the citrus viroids. hybridization with other viroids. Extracts from tissue infected with BIOLOGICAL PROPERTIES selected isolates (LiCl supernatant samples or CFll chromatographed Transmission to herbaceous preparations) were denatured (311, hosts. Extracts from selected isolates applied to nitrocellulose membranes derived from field sources were slash using an Hibri-slot filtration manifold inoculated to Gynura aurantiaca DC, (BRL), and hybridized to cDNA made tomato, eggplant, Gomphrena globosa to CEV and CV-Ib, as well as to ASV L. and Datura stramonium L.. Gy- (a non-citrus viroid control). The nura and tomato plants inoculated cDNA was synthesized by the method with CEV-containing isolates showed of Taylor et al., (26). The reactions stunting, epinasty and leaf rugosity were dependent on the viroid profile characteristic of CEV infection 3 to 4 of the isolate and the cDNA probe weeks after inoculation, whereas the utilized. As summarized in table 1, other species were symptomless. In- cDNA to CEV-RNA showed a strong oculation with isolates which did not hybridization reaction only with prep- contain CEV, did not induce Diseases Induced by Viroids and Viroidlike Pathogens

Fig. 2. Autoradiographs of slot-blot hybridizations on nitrocellulose membranes of nucleic acid preparations of citron and Gynura inoculated with different exocortis isolates using a cDNA to citrus exocortis viroid (CEV) probe. In all instances 10 p1 samples were applied using a Hybri-slot filtration manifold. a) Samples were 2M LiCl soluble nucleic acids of CEV containing isolates: El17 and El29 on Gynura (1 and 2), and E117, E123, E104, E129, El24 and El28 on citron (3 to 9). b) Samples were 2M LiCl soluble nucleic acid preparations of mild isolates: E125, E122, E147, E146, E152, E151, El53 (1 to 7), pure CV-Ia (8) and CV-IIa (9) on citron. c-d) Samples were nucleic acid preparations after CF-11 cellulose chromatography of healthy (1) and isolates E807, E804, E819, E821 , E803, E820, E812, E814 (2 to 9) on citron. Eluants at 25% EtOH (d) and DO% EtOH (c) of the same preparations were processed simultaneously.

symptoms in any of the five species. were pruned three weeks after inocu- All inoculated plants were pruned lation. When the second growth flush four weeks after inoculation. The see- emerged characteristic symptoms ond growth flush of tissue was col- were observed on plants inoculated lected and the nucleic acids extracted with CV-11-containing sources. Both as before and analyzed by sequential CV-IIa and CV-IIb (25) induced gel electrophoresis. A CEV band was darker color, shorter internodes, observed in all hosts whether smaller leaves with rugose leaf blades symptomatic and symptomless, and distorted fruits. When the nucleic whereas CV-I1 was detected only in acids were extracted and analyzed by eggplant (fig. 3). The CV-11-contain- sequential gel electrophoresis CV-IIa ing eggplants were symptomless. and CV-IIb were detected from Cucumber seedlings of the variety symptomatic plants. A CEV band 'Suyo' (kindly provided by Sakata could also be detected from symptom- Seed Corp.) were inoculated by stem less plants which had been inoculated puncture when the first true leaves with CEV-containing isolates (fig. 3). were just emerging. All inoculated The infectivity of CV-IV was cucumbers were symptomless and tested only on Gynura and cucumber. Tenth ZOCV Conference

Fig. 3. Polyacrylamide gel electrophoresis (PAGE) of nucleic acid extracts under low pH, denaturing conditions (8M urea) following electrophoresis in 5% gels as described as sequential PAGE. Samples were from healthy and inoculated cucumber, eggplant, gynura, tomato, Gom- phrena and Datura. An electrophoretic profile of isolate El17 from citron is shown as a reference for migration of the different citrus viroids. Gels were silver stained.

This viroid was recovered from tions have been produced by inocula- symptomless inoculated cucumber tion with nucleic acid preparations plants (table 3). from herbaceous hosts (CEV, CV-I1 Therefore, in addition to the de- and CV-IV) or by electroelution of scribed differences in physical proper- single bands obtained after perform- ties, citrus viroids have quite differ- ing sequential PAGE (CV-I and CV- ent host ranges. Whereas CEV can 111). Single viroid sources have also be recovered from a large number of been obtained by shoot tip grafting in herbaceous hosts (30) and induces vitro (see next section). characteristic symptoms in several of Inoculations of citrons with pure them, CV-I1 and CV-IV have a more CEV preparations caused severe restricted host range. No host outside stunting, leaf epinasty and necrosis the Rutaceae has yet been found for (fig. 4A and 4B). Differences in CV-I and CV-111. Therefore, citron symptom intensity have been found remains the common host from which among different samples and are all described citrus viroids can be eas- thought to be caused by minor differ- ily recovered. ences in the nucleotide sequence and Symptomatology in citron. Al- in the number of nucleotide residues, though citrus viroids are usually as described by Visvader and Symons found as mixtures, single viroid infec- (28). Citron plants inoculated with Diseases Induced by Viroids and Viroidlike Pathogens 159

TABLE 3 BIOLOGICAL PROPERTIES OF CITRUS VIROIDS

Infectivity Symptoms in Citron Citrus midvein leaf petiole leaf tip viroids Gynura Cucumber Citron stunting necrosis epinasty necrosis browning

CEV + + t severe general severe general ? + local random - - local random - - none - - - faint none - - - - a local random - - local random - - local general - - mild general general -

local random - - preparations containing CV-Ia or CV- symptomatology on citrons has not Ib showed a pronounced epinasty or yet been made. Inoculation of citrons bending of the leaves because of point with pure CV-IV resulted in random necrosis of the midrib on the under- leaf epinasty and midvein necrosis side of the leaf blade (fig. 4B and 4D). (table 3). This symptom occurs in a random Comparison of the symptoms in fashion and affects only a few leaves citron produced by single viroids with of the inoculated plants. In general, those produced by mixed infections citrons inoculated with pure sources indicates existence of synergistic vir- of CV-IIa or CV-IIb were symptom- oid interactions. The E821 isolate, less. However, E818 and E819, which originally described as producing are naturally occurring pure sources moderately severe symptoms, under of CVC-IIa, produced a mild response optimal environmental conditions when inoculated on Arizona 861-S1 caused symptoms in citron similar to citron under optimum conditions of those induced by CEV, even though temperature and day length. The CEV is not a component of this com- reaction was a faint necrosis of the tip plex isolate. Under different environ- of the leaf blade (fig. 4G) in a small mental conditions (18), a variable number of leaves and sometimes a reaction with periodic production of very mild petiole wrinkle and occa- symptomless and symptom-expres- sional petiole browning. All sources of sing growth was evident. These CV-IIb failed to show symptoms on symptoms were more severe than ex- citrons incubated under a range of en- pected from a combination of the vironmental conditions. symptoms induced by each compo- The symptoms induced by CV-I11 nent poresent. Also, the variable na- were characterized by different de- ture of the citron response suggest grees of petiole wrinkle and necrosis that different components of the vir- (fig. 4E) accompanied by midvein ne- oid complement predominate under crosis which sometimes affected sec- different environmental conditions. ondary veins. This resulted in a Similarly, the mild symptoms as- marked leaf epinasty which was gen- sociated with both CV-IIIa and CV- eral (fig. 4C), or randomly affected IV look unrelated to the moderate only some leaves (fig. 4F). Some de- symptoms induced by the original iso- grees of mild stunting also developed. lated E804 which contains both vir- A comparative study to determine oids. whether the different CV-I11 group Artificial viroid mixtures. The viroids can be discriminated by majority of the "exocortis" isolates Tenth ZOCV Conferenee Diseases Induced by Viroids and Viroidlike Pathogens 161 studied were found to occur as viroid combination of two viroids (table 4). mixtures (table 2). Although the The production of stable combina- number of isolates described does not tions and single viroids suggests that include all possible viroid combina- all the viroids tested here can be ob- tions, artificial mixtures have been tained as pure isolates and all viroid obtained and also shown to be stable. combinations should be possible. In In fact, the recovery of stable, fact, attempts to obtain viroid combi- mild forms of "exocortis" by knife nations by mixed inoculations have blade transmission from severely af- been successful (table 4). fected citron donors was already reported in 1969 (15). Although the pro- HORTICULTURAL cess was not understood at that time, IMPLICATIONS the possibility of several strains of the disease existing as components of a During the early studies on the disease complex was suggested. Iso- transmission of the exocortis disease, lates E806, E811 and E812 from severe stunting, epinasty and leaf California were recovered by knife rugosity induced on inoculated citrons transmission from a single donor were associated with severe dwarfing (E800). These were originated by in- and bark scaling observed in field dependent transmission of the viroid trees grown on sensitive rootstocks components present in the original (2, 5,14). Also, a large number of mild source which resulted in two single and moderate sources identified ac- viroid isolates and one isolate contain- cording to their reaction on citron ing two viroids (table 2). were detected during routine index- Other artificial viroid profiles have ing of symptomless field trees grown also been produced by greenhouse on sour orange and other symptom- and laboratory manipulations (table less rootstocks. Therefore, the pre- 4). The differences in transmission cise relationship between the citron properties of the different viroids can reaction and the overall disease syn- be utilized to segregate some of the drome in the field remains unclear. components present in a complex iso- With this present understanding, late. In fact, Gynura and tomato, it appears that the "exocortis dis- which have long been utilized as pri- ease", as has been developed in the mary hosts for viroid purification, literature, constitutes a complex of also functioned as selective hosts for apparently unrelated viroids, some of CEV. The availability of cucumber which may not produce the classical and eggplant as screening hosts for symptom of the disease on trifoliate CV-I1 should help in the identification orange. In addition, the availability of and characterization of members of pure viroid sources will provide a bet- this viroid group. ter understanding of the described The application of shoot-tip graft- "dwarfing factors". Perhaps a viroid ing in vitro (12) to Arizona 861-S1 cit- responsible for dwarfing alone could rons infected with the Spanish isolate be safely separated from deleterious El17 also resulted in two single viroid mixtures or engineered from labora- sources and a source containing a tory produced mixtures. * Fig. 4. Symptomatology in citron after inoculation with CEV and citrus viroids (CV). A) Severe stunting and epinsaty induced by pure CEV. B) Bending of the leaves as a result of point necrosis of the midrib on the underside of the leaf blade (see arrow) induced by pure CV-Ia. C) Leaf epinasty as a result of petiole bending induced by pure CV-IIIc. D) Leaf symptoms induced by pure CEV (right), pure CV-Ia (middle right), pure CV-IIIc (middle left) and healthy control (left). E) Healthy control (right), and petiole wrinkle (middle) and necrosis (left) induced by pure CV-IIIc. F) Symptoms induced as a result of a mixed inoculation (CV-Ia, CV-IIa and CV-IIId). G) Faint necrosis of the top of the leaf blade (see arrow) induced by CV-IIa (right) and healthy control (left). 162 Tenth ZOCV Conference

TABLE 4 VIROID PROFILES OBTAINED THROUGH LABORATORY AND GREENHOUSE MANIPU- LATION

Viroid Profile Isolate Technique origin CEV Ia Ib IIa IIb IIIa IIIb IIIc IIId IV

Screening host: Gynura ‘‘severe7TZ + ------Cucumber E 144 - - - +------Cucumber E821 - - - ++----- Cucumber Ego4 ------+

Selective removal: Shoot tip grafting E 117 - + ------E 117 ------+ - E 117 - +------+ - E 117 - + - +------Selective transmission: Inoculation El17 + - - +------E 117 - + - +------El17 + + - +------El17 + - - +---- + - E 117 - + - +---- + - "Citrus exocortis viroid (CEV) can be recovered froni Gynura inoculated with all "severe" isolates.

In order to define the cause-effect and persistence of this reaction in the relationship between the different cit- field trees must be evaluated. rus viroids and the "exocortis dis- A citron reaction to CV-IIa has ease", it will be necessary to study not been consistently observed. In the performance of trees inoculated many instances citrons inoculated with single viroid and specific viroid with CV-IIa were symptomless, and combinations. The results of this test- an Arizona 861-S1 citron line main- ing will identify the potential disease tained at IVIA as a healthy source causing agents and their relationship was found to carry CV-IIa. There- to the exocortis disease as originally fore, one should exercise caution defined. when using citron as the sole indicator Some preliminary results are al- for citrus viroids. ready available from inoculations made in 1977 and 1983 with field iso- CONCLUSIONS lates from California. These results il- lustrate the significant stunting effect The data presented here on the of CEV on trees on Troyer citrange role of several viroids on the develop- as well as on sour orange whereas the ment of symptoms in citron, which other citrus viroids had little effect on were originally thought to be as- trees grown on the same rootstocks sociated with the exocortis disease, (11). However, sweet orange trees provides the basis for a new under- grown on trifoliate orange which were standing of the disease and its causal inoculated with E818 and E819 agents. (naturally occurring pure sources of The study of a number of naturally CV-IIa) in 1983 were already showing occurring field isolates, and the deter- the first symptoms of bark cracking. mination of the physical and biological Therefore, CV-IIa the viroid causing properties of their viroid components the mildest symptoms on citron in- permits the cataloguing of citrus vir- duced the classical symptoms as- oids into five different groups. Al- sociated with the exocortis disease in though ten viroids from California the field. The further development and Spain, with different elec- Diseases Induced by Viroids and Viroidlike Pathogens 163 trophoretic mobilities have been used and even larger number of viroids as the basis for a consensus catalogue with distinct properties. of citrus viroids, it is likely that the ACKNOWLEDGMENTS analysis of other isolates in different citrus growing areas of the world will The authors would like to thank result in further elaboration of this Dr. Luis Navarro for his helpful com- grouping. Studies of the physical and meents during the development of the biological properties of these isolates project, and Juana Maria Perez and will probably reveal the existence of Maria Isabel Molins for their technical assistance.

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