Details of DPV and References

DPV NO: 226 September 1980

Family: Pospiviroidae
Genus: Pospiviroid
Species: Citrus exocortis viroid | Acronym: CEVd

Citrus exocortis viroid

J. S. Semancik Department of Plant Pathology and Cell Interaction Group, University of California, Riverside, California 92521, USA

Contents

Introduction

Disease described by Fawcett & Klotz (1948) and transmission described by Benton et al. (1950).

The infective agent is a small naked single-stranded RNA (c. 105 daltons) which associates with host nucleus and membranes. There is no evidence for vector transmission. The agent is stable in sap, resistant to thermal inactivation and easily transmitted mechanically. It infects many species in the Rutaceae and a few in the Solanaceae and Compositae. Widespread in citrus-growing regions of the world.

Main Diseases

In citrus the agent causes shelling, scaling and splitting of the bark of susceptible rootstocks (Fig. 1), hence the name ‘exocortis’ (exo = outside, cortis = pertaining to the bark; Bitters, 1981). Other names for the disease are ‘scaly butt’ (Benton et al. 1949) and ‘Rangpur lime disease’ (Olson, 1952). Trees on susceptible rootstocks are stunted and yield poorly (Fig. 4). Leaf epinasty and severe stunting is common in indicator species (Weathers, Greer & Harjung, 1967).

Geographical Distribution

The disease is present in most citrus-growing areas where susceptible rootstock is used. It is widespread in S. America (especially Brazil and Argentina), Australia and the Mediterranean region (especially Spain) but is of limited occurrence in the USA and N. Africa. Commercial plantings in Japan are essentially free from infection (Wallace, 1978).

Host Range and Symptomatology

The host range is largely restricted to the citrus family (Rutaceae) although some species in the Solanaceae (Solanum tuberosum, Lycopersicon esculentum, Petunia hybrida) and Compositae (Gynura aurantiaca, G. saramentosa) are susceptible. Transmitted easily by mechanical inoculation of sap from Gynura (Weathers & Greer, 1968) but with difficulty from Citrus (Garnsey & Jones, 1967).

Diagnostic species

Citrus sinensis (sweet orange) on Poncirus trifoliata (trifoliate orange) rootstock. Between 1 and 2 years after graft inoculation the plants show classical symptoms of bark shelling and scaling of the rootstock and a decrease in tree vigour leading to stunting and yield reduction.

Citrus medica (Etrog citron). Leaf epinasty and rugosity (Fig. 2, Fig. 3) cracking and browning of the underside of the veins and severe stunting visible from 3 weeks to 6 months after graft or razor slash inoculation. There are preferred sensitive clones such as Arizona 861 or USDCS 60-13 (Calavan et al., 1964).

A wide range of citrus species and varieties respond but differ in the time of appearance and severity of symptoms.

Gynura aurantiaca (velvet plant). Principal herbaceous indicator (Fig. 6). The symptoms are similar to those in Etrog citron but are visible from 10 to 30 days after inoculation with sap (Weathers & Greer, 1968).

Lycopersicon esculentum (tomato) cv. Rutgers. Same symptons as Etrog citron; visible from 10 to 30 days after inoculation with sap (Semancik & Weathers, 1972c).

Propagation species

Gynura aurantiaca or Lycopersicon esculentum. Citrus medica (Etrog citron) is a good host for some field isolates that cannot be transmitted to herbaceous test plants.

Assay species

Citrus medica (Etrog citron) and Gynura aurantiaca.

Strains

Differences among field isolates in the severity of symptoms induced, incubation period and degree of stunting were attributed to the existence of strains (Fraser & Leavitt, 1959; Rossetti, 1961; Calavan & Weathers, 1961; Weathers & Calavan, 1961). It has been suggested that the mild strains occur in leaves (Pujol, 1965) and cause stunting whereas the severe strains cause bark shelling (Vogel, Bové & Bové, 1965). No evidence exists for cross protection between mild and severe strains (E. C. Calavan, unpublished data; J. S. Semancik & L. G. Weathers, unpublished data).

Transmission by Vectors

No evidence for transmission by a vector.

Transmission through Seed

Tests for seed transmission in citrus have been negative (Bitters, Brusca & Dukeshire, 1954) except for a single report (Salibe & Moreira, 1965) which might be reinterpreted as natural spread. The viroid can be transmitted through the seed of Lycopersicon esculentum cv. Rutgers (J. S. Semancik, unpublished data).

Transmission by Dodder

Transmission from citrus to citrus and from citrus to petunia was accomplished by means of Cuscuta subinclusa (Weathers, 1965a, 1965b).

Serology

No positive application of serological techniques has been reported. The naked-RNA structures of the pathogen would not be expected to be immunogenic, and as no viroid-specified proteins have been detected (Hall et al., 1974) any serological reaction could only result from the detection of an increase in a host-specified protein (Conejero & Semancik, 1977).

Relationships

‘Strains’ in citrus have been recognized on the basis of severity of symptoms on various rootstock/scion combinations. No physical description of these isolates nor any evidence for cross protection in citrus has been reported. Symptoms in tomato and potato (Semancik, Magnuson & Weathers, 1973) are identical to those caused by potato spindle tuber viroid. The nucleotide sequences of the two viroids are nevertheless distinct (Dickson, Prensky & Robertson, 1975; Gross, Domdey & Sänger, 1977). Cross protection, defined as ‘an interference in symptom expression’, was reported (Niblett et al., 1978) when the two viroids were inoculated to tomato. However, when buds from citron with the severe strain of citrus exocortis viroid were grafted to citron containing the moderate strain, the severe strain became predominant, indicating no cross protection (J. S. Semancik & L. G. Weathers, unpublished data). The similarity in physical properties and in symptoms (stunting and epinasty) of all viroids suggests that there are close biological relationships among these small pathogenic RNA species.

Stability in Sap

The thermal inactivation point (10 min) is 90-100°C in sap of G. aurantiaca and even higher in partially purified preparations of viroid RNA. The agent remains infective on a contaminated cutting blade for at least 16 h (Garnsey, 1968).

Purification

The agent can be recovered for biological tests or physical characterization by employing techniques for purification of nucleic acids. Purified preparations are highly infective. The commonest procedure is to blend infected leaves in a two-phase phenol-buffer emulsion and precipitate the RNA from the aqueous phase with 75% ethanol. The material soluble in 2 M LiCl is then subjected to polyacrylamide gel electrophoresis (Fig. 5) (Semancik & Weathers, 1972b; Semancik et al., 1975).

Properties of Infective Nucleic Acid

The infective agent is a small RNA of about 1.1 to 1.2 x 105 daltons (Semancik & Weathers, 1970, 1972a; Semancik et al., 1975; Sänger et al., 1976). The molecule is single-stranded even though it elutes from methylated albumin and CF-11 cellulose in a manner characteristic of double-stranded RNA. Partial phosphodiesterase resistance (Semancik & Weathers, 1970) suggests that the RNA has a circular structure similar to those of other viroids. Nucleotide composition of G 28.8: A 21.5: C 29.4: U 19.9 along with a Tm = 52°C and 27% hyperchromicity in 0.15 M NaCl + 0.015 M Na citrate indicates a GC-rich molecule with a high degree of GC base pairing.

Low field nuclear magnetic resonance spectrum, solubility in 2 M LiCl, as well as resistance to inactivation by diethylpyrocarbonate further suggest a highly structured self-complementing molecule. The buoyant density in Cs2SO4 is about 1.64 to 1.68 g/cm3 and s20,w = 6.7.

Relations with Cells and Tissues

The agent can be isolated from all plant parts including roots and fruit (Semancik, Grill & Civerolo, 1978). Regions of meristematic activity such as shoot apices or neoplastic tissue contain a high concentration of the viroid RNA. Symptoms suggesting vascular disorders may indicate association with vascular tissues. The subcellular distribution of the agent indicates that it is localized in the nucleus and endomembrane complex (Semancik et al., 1976). The only subcellular cytopathic change observed is an increase in plasmalemmasomes, or membraneous endocytic vesicles in the paramural area (Semancik & Vanderwoude, 1976).

Acknowledgements

The author wishes to recognize the valuable suggestions and consultations of Professors W. Bitters, E. C. Calavan and L. G. Weathers in the preparation of this description.

Figures

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Bark scaling symptoms of exocortis disease on Morton citrange rootstock. (Courtesy of L. G. Weathers.)

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Foliar symptoms on Etrog citron caused by severe isolate (left) and moderate isolate (right) about 6 weeks post inoculation.

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(Left) Stunting and severe epinasty of Etrog citron, (right) healthy plant. (Courtesy of L. G. Weathers.)

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Field symptoms. The tree on the left shows general stunting and reduced vigour 8 years after infection with the exocortis disease agent. The tree on the right is not infected. (Courtesy of D. J. Gumpf.)

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Electrophoresis (from left to right) on 5% polyacrylamide slab gel of RNA extracts from tomato, (a) healthy, (b) infected with citrus exocortis viroid, (c) as (b) but treated with DNase. Stained with ethidium bromide.

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Gynura aurantiaca, (left) 8 weeks post inoculation with a severe isolate, (right) uninoculated control.

References list for DPV: Citrus exocortis viroid (226)

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  2. Benton, Bowman, Fraser & Kebby, Sci. Bull. Dep. Agric. N. S. W. 70: 20 pp., 1950.
  3. Bitters, in The Citrus Industry, Vol. V, eds. W. Reuther, E. C. Calavan & G. E. Carmen, Berkeley: Univ. Calif. Div. Agric. Sci., 1981.
  4. Bitters, Brusca & Dukeshire, Citrus Leaves 34: 8, 1954.
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