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DESCRIPTION AND BACKGROUND
The exocortis disease of citrus, caused by the citrus exocortis viroid (CEV), was first reported and described by Fawcett and Klotz (1948) as a bark-shelling disorder of trifoliate orange rootstock. Benton et al. (1949) reported that this disease had been known in Australia since the early 1930s as "scaly butt" and had been found to be transmissible. They recommended that budwood for propagation be taken only from older trees on trifoliate rootstock showing no scaling of the butt or trunk.
The disease is present in almost all citrus-growing regions of the world. Although many of the commercial citrus cultivars are symptomless carriers, trees may be stunted to some degree on rootstocks normally considered tolerant. In countries where trifoliate orange is the primary rootstock, bud selection over many years has avoided CEV, but other citrus viroids may be present.
Shell bark of lemon is sometimes confused with exocortis. Although CEV or a citrus viroid (CV) may be factors in enhancing shell bark symptoms, they may not be causal. Incidence of shell bark is greatly diminished by use of virus and viroid-free nucellar or shoot-tip grafted lemon bud selections.
Gummy pitting and gum pocket have been reported from Australia and South Africa and may be associated with certain citrus viroids. Symptoms are gum-impregnated pits or pockets in the trifoliate rootstock, which can be seen on the wood surface if the bark is removed. Gum impregnated areas range from few to numerous and from small to large (Fraser and Broadbent,1979) and are more prevalent in older trees.
As stated above the causal organism of exocortis disease is the citrus exocortis viroid (Semancik and Conejero-Tomas,1987; Semancik,1988). It is a low-molecular-weight RNA consisting of 371 nucleotides. It can exist as either linear or circular molecules and is highly mechanically transmissible by tools from tree to tree. CEV is transmissible into Gynura aurantiaca, , petunia or tomato plants, and causes distinct and characteristic severe epinasty symptoms on leaves of these hosts. Nucleic acid extraction can be made from these hosts or from young symptomatic citron shoots and analysed by polyacrylamide gel electrophoresis (PAGE). CEV will migrate in a band on the gel and can be visualized by staining with silver or ethidium bromides.
Recent studies indicate that there are a number of citrus viroids of a molecular weight lower than CEV that can also induce symptoms in citron. Some of these viroids induce mild bark cracking in trifoliate orange (Figure 37) distinct from the severe bark shelling associated with CEV. These citrus viroids should be considered as independently transmitted and distinct pathogens. Schlemmer, Roistacher and Semancik (1985) were the first to report that citron-variable viroid reacted only in citron but not in Gynura or other herbaceous hosts in which CEV reacts and multiplies. Duran-Vila et al. (1988) reported on four additional distinct viroid groups (named citrus viroids I to IV) found in Spain and California, with a size range down to 275 nucleotides. These viroids induced specific mild reactions in citron, and were found in field trees of citrus in pure form or in various combinations. Currently there are some ten to 12 viroids in the CV complex, some of which appear closely related, based on nucleotide number and nucleic acid hybridization assays. However, they may induce different reactions in field trees. For example, the cachexia viroid will migrate on a polyacrylamide gel and form a band (IIb) containing approximately 300 nucleotides. It very closely resembles another citrus viroid (IIa) that contains about 305 nucleotides. However, symptoms induced in citron and other citrus cultivars and rootstocks are clearly and strikingly different. In any indexing programme (citrus, grape or stone fruit) with its object of producing disease-free primary stock, it is important that all viroids be recognized and, if possible, removed from propagative budwood.
Citrus viroids are distributed primarily by the introduction and propagation of infected budwood and subsequently by mechanical transmission. The viroids are transmitted mechanically by hedging equipment, tools and knives, especially from lemon to lemon. Mechanical transmission of CEV was first demonstrated by Garnsey and Jones (1967), who showed that contaminated tools could be disinfected by a mixture of 2 percent sodium hydroxide plus 2 percent formaldehyde. Roistacher, Calavan and Blue (1969) demonstrated that low dilutions of sodium hypochlorite are readily available, less toxic and a very efficient disinfectant for CEV. Although mechanical transmission from orange, mandarin or grapefruit is less efficient than from lemon (Garnsey,1968), once the viroid is present it will spread from tree to tree throughout an orchard over a period of time by hedging, pruning, clipping of fruit or collecting of budwood. Neither CEV nor CVs are known to be vector- or seed transmitted, and root transmission, though possible, would be overshadowed by mechanical transmission.
All of the citrus viroids appear to be readily eliminated by shoot-tip grafting or by use of nucellar budlines. They are extremely tolerant to heat and have not been successfully eliminated from budwood by thermotherapy.
At first the diagnosis of exocortis was a long-term process in which trifoliate orange or Rangpur lime as a rootstock, under a vigorous growing scion such as lemon, was inoculated and placed in the field. Symptoms would appear in the rootstock after two to six years or longer, depending on the severity of the viroids present. The use of citron as a rapid indicator was first proposed by Salibe and Moreira (1965), tested by Frolich et al. (1965), and modified by Calavan et al. (1964), who demonstrated rapid development of symptoms by forcing a bud of a sensitive clonal line of citron on a vigorous seedling stock. Roistacher et al. (1977) developed a citron selection via shoot-tip grafting (861-S-I), which is highly sensitive to CEV and the milder reacting CVs.
The detection of CEV and related CVs by graft-transmission and by PAGE requires the production of the highest quality plants growing under ideal conditions of nutrition and temperature. The need for an exacting type of plant laboratory (as outlined in Part II) becomes apparent when indexing for these viroid-induced diseases. Recent important developments in the detection of a whole range of new citrus viroids are reviewed by Duran-Vila et al. (1988) and techniques for PAGE analysis are given in Part III. Principles relating to these viroids may be applicable to similar viroids now being found in grapes and stone fruit.
Field symptoms range from mild bark cracking to very severe bark scaling, primarily on trifoliate and Rangpur lime rootstocks, accompanied by various degrees of stunting of the tree (see Figures 35-37 and 43). For more detailed descriptions and photographs see Weathers (1980) and Wallace (1978). Both Rangpur lime and trifoliate orange twigs and branches may show a chlorotic stem blotching which is symptomatic (Figure 38). Citrons are highly susceptible and, when inoculated, may show bark cracking (Figure 41b), leaf epinasty and necrosis of the leaf veins (Figure 41a). Sweet limes and certain lemon varieties may show elongated bark cracks. Certain mechanically inoculated herbaceous hosts, i.e. Gynura, petunia and tomato, will show a distinct leaf curl, epinasty and vein necrosis. Trees on trifoliate hybrid rootstocks can be stunted to varying degrees (Figure 43) and some of these hybrid rootstocks will show bark cracking. Trees on trifoliate rootstock affected with both exocortis and tristeza can be very severely stunted. Trifoliate orange rootstock infected with individual or combinations of CVs (excluding CEV) may show mild to moderate bark cracking (Figure 37). Occasionally CEV can induce stunted, acorn-shaped fruits on citron or Volkamer lemon (Figure 39) (Bitters, Duran-Vila and Semancik,1987).
METHODS OF DETECTION Method 1: Field diagnosis
If the rootstock is trifoliate orange, certain citranges or Rangpur lime, typical bark cracking as shown in Figures 35 and 36 is symptomatic and diagnostic for CEV. Citrus viroids I to IV (excluding cachexia) do not induce the severe bark cracking typical of CEV, but may cause a mild bark cracking in trifoliate orange stock (Figure 37).Twigs and branches of CEV-infected trifoliate orange or Rangpur lime may show a yellow blotch as seen in Figure 38. The use of trifoliate orange as a seedling or rootstock for long-term field indexing is no longer recommended. However, it may be useful for detecting and classifying the milder reacting CVs. Studies under way show that certain CVs will crack trifoliate orange rootstock in the field and cracking will vary, depending upon combinations of viroids present in the inoculum (Roistacher and Semancik, unpublished). Severe stunting of trees on trifoliate orange rootstock, even in the absence of bark scaling, may indicate a viroid infection.
Method 2: Citron index
The use of sensitive clonal citron selections such as 60-13, 861 or 861-S-I budded to a vigorous rootstock such as rough or Volkamer lemon, or propagated as cuttings, is the preferred and recommended index method. It is relatively rapid, sensitive and highly diagnostic (Caravan et al.,1964; Roistacher et al.,1977). Citron seedlings may be used for detection of CEV, which causes strong symptoms. However, when indexing for the milder reacting citron viroids of groups I to IV, 861-S-I budded to a vigorous rootstock is the recommended indicator.
Collection of budwood. In a routine index, a minimum of four budsticks should be collected, one from each quadrant of the tree. However, for periodic critical reindexing of foundation block trees, where they may have been subject to possible contamination by infected tools via mechanical transmission, eight budsticks should be collected from around the tree. In the Citrus Variety Improvement Program in California, each foundation tree is indexed once every three years for possible contamination by CEV or CVs. At times CEV infection has been discovered in only one of the eight sectors of the tree. Whenever collecting budwood, tools must be dipped or sprayed with a 1 percent sodium hypochlorite solution when going from tree to tree.
This practice is extremely important and necessary as a sanitary precaution, and should be incorporated as standard sanitary procedure in the field as well as in the plant laboratory.
Inoculum tissue. The best inoculum tissue for indexing is the "bud" (bud, blind bud or chip bud). A minimum of two inoculum buds are recommended per test plant. Leaf tissue should not be used. Studies by Blue et al. (1975) showed poor transmission of CEV by leaf-disc grafts.
Inoculation. The preferred indicator scion is 861-S-1. When obtained, a bud of 861-S-1 is propagated on rough lemon or other lemon type stock and maintained as a source plant. Extreme care should be taken to disinfect clippers when collecting budsticks from the source plant for use as scion indicators. A 1 percent sodium hypochlorite solution is the preferred disinfectant for CEV or other CVs (Roistacher, Calavan and Blue,1969).
The 861-S-1 citron bud is then grafted to a vigorous seedling rootstock, such as rough or Volkamer lemon, at about 25 cm above the soil surface. (Other rootstocks should be tested for compatibility, since citron does not grow well on all rootstocks.) When wrapping the citron bud with budding tape, the "eye" of the bud may be exposed for forcing, or it can be completely wrapped, and unwrapped two or three weeks after budding.
Inoculation can be done at the same time as grafting the citron scion bud. Two inoculum "buds" are grafted anywhere below the citron bud and completely wrapped. The seedling is then bent just above the citron bud and the top portion of the seedling tied to the base of the plant to aid in the forcing of the citron bud (Nauer and Goodale,1964). This is the same procedure used for forcing the Parson's Special mandarin bud in the cachexia index (Cachexia
Figures 47 and 48). However, if rough lemon seedlings are used as the rootstock, the seedlings can first be cut back at the time of inoculation to 25 or 30 cm above the soil surface and the citron bud inserted near the top of the cut seedling and wrapped. The inoculum buds are then inserted below and wrapped. The compatibility of citron with rough lemon is excellent and cutting back at the time of inoculation favours the forcing of the citron bud. This procedure is more convenient than bending and has been successfully used.
Number of indicator plants. Indicator plants should be grown one per container for CEV indexing. The milder symptoms associated with citrus viroids are best expressed and observed in plants grown one per container. A minimum of four inoculated plants is recommended for each index test, and the inoculum collected from the various sectors of the field tree to be tested should be uniformly distributed among the test plants. After inoculation, the used inoculum budwood should be refrigerated and saved. It can be used later to reinoculate any plants where grafts have failed, provided that fresh inoculum is not readily available.
Controls. Two positive controls are sufficient for CEV. However, the mild-reacting citrus viroids (Duran-Vila et al.,1986,1988) require additional specific positive mild controls for their identification. The very mild-reacting citrus viroids such as CV IIa require more control plants (six or eight are recommended). The mild-reacting citrus viroids are usually found in mixtures with CEV and can be isolated from CEV by mechanical transmission or by shoot-tip grafting (Roistacher et al.,1969; Duran-Vila et al.,1988). When found, they can be preserved by transferring them into sweet orange seedlings and holding these as source plants in a "virus" bank. The specific type and identity of the viroid can be determined by PAGE (Part III).
Inoculum survival. After two or three weeks the budding tapes covering the inoculum "buds" plus the citron scion bud should be removed. It is extremely important that the razor-blade or knife used to cut the tape be disinfected in a 1 percent sodium hypochlorite solution when moving from plant to plant. Any dead or dying grafts should be recorded and the plant reinoculated, or a new test plant used if both inoculum grafts are dead. If the citron scion bud at the top is dead, the plant should be rebudded or a new test plant established.
Post-inoculation care. The forced citron scion bud should be trained to grow as a single shoot or leader, as illustrated in Figures 47 and 48. The rootstock stub should be cut close to the emerging citron shoot and clippers must be disinfected between each cut. Citron has a natural tendency to grow as a single shoot in the plant laboratory, and only occasional suckering or trimming of side shoots is needed. The young shoots or suckers should be removed by pulling them off and not rubbing them off. Exocortis can be spread from plant to plant on the hands after rubbing off sucker shoots (Roistacher et al., 1969). When the young growing shoot is approximately 10-15 cm long, it should be tied to a stake as shown in Figure 48.. Standard bamboo stakes are satisfactory but should be sterilized before use to prevent contamination of the soil by pathogenic organisms. Plants should be well spaced on the bench, leaving about 400 sq cm (about 20 x 20 cm) per plant (Nauer, Holmes and Boswell,1980). Allow the plants to reach 1 m or more. If symptoms do not show in the leaves of the mild positive controls, perhaps owing to insufficiently warm growing conditions? cut the plants back at about 10 cm above the bud-union. Retrain the new shoot to a single leader as before and once again observe new growth for symptoms.
Temperature requirements. The importance of maintaining warm or hot growing temperatures for maximum symptom development of CEV or CVs cannot be over-emphasized. Recommended temperatures are 32-40°C maximum during the day and 27-30°C minimum at night. Avoid temperatures above 40°C since leaves may become distorted and small (Reuther, Nauer and Roistacher,1979). Figure 40 illustrates the importance of temperature for symptom expression in citron. Both plants were inoculated with certain citrus viroids. The plant on the let's shows no symptoms and was held at normalcool maximum day temperatures of 25-30°C, whereas the plant on the right showing symptoms was held at maximum day temperatures of 32-40°C. The inoculum was from a moderate-reacting citrus viroid.
Time for first symptom development. The severe symptoms associated with CEV generally appear in four to ten weeks when citrons are grown under warm conditions as single shoots. The mild symptoms associated with other citrus viroids take considerably longer, i.e. three to six months. Positive controls should be continually observed for tip browning, petiole browning, petiole wrinkle and mild leaf epinasty (Figure 42). These will occur only under proper conditions of nutrition, temperature and proper salinity balance. Non-inoculated plants to serve as negative controls are essential.
Symptoms. The classic leaf-epinasty symptom for CEV in citron is seen in Figure 41a. The underside of the leaves will show brown, necrotic and cracked veins, especially in the midvein. The petiole will be severely wrinkled or cracked and discoloured. The bark of the stem may be severely cracked as in Figure 41 b. Cracking may be severe to mild depending on the strain and environment.
Leaf symptoms for the milder-reacting citrus viroids are tip browning (Figure 42a), petiole wrinkle and browning (Figure 42b), midvein browning (Figure 42c), and mild leaf epinasty (Figure 42d; also Roistacher et al.,1977). Petiole wrinkle may occur without browning. However, as the plant matures, browning of the petioles on the lower leaves becomes more pronounced and distinct. Leaves of the non-inoculated control plants grown with proper nutrition, temperature and watering will show no petiole wrinkle or browning (Figure 42b, right). As mentioned earlier, these symptoms are very mild and will be seen only under the best conditions of growth and temperature. They will not be seen if temperatures are too cool (Figure 40). They may not be seen in seedlings, and seedlings should not be used for detection of these milder-reacting citrus viroids (Roistacher et al.,1977).
Termination. The index can be terminated when most or all of the mild-positive controls show clear, definitive, positive reactions. If no reaction is evident in the leaves of the mild-positive control plants after they have grown to over 1 m as single shoots, all plants should be cut back to about 10 cm above the bud-union, the temperature in the greenhouse may need to be raised, and a new citron bud forced and grown as a single shoot. The leaves on this new shoot should be observed and compared with those on negative control plants until definitive symptoms are evident.
MISCELLANEOUS
Seedling index
On occasion, seedlings of 861 citron may be used if CEV is the only viroid under index (Garnsey and Whidden,1973); they should not be used if the milder-reacting citrus viroids are to be indexed. The procedures using seedlings are similar to those outlined in Method 2. Two to four indicator plants can be used. Positive and negative controls should be included and seedlings can be cut back at the time of inoculation. Seedlings should be grown one per container and trained as single shoots. Temperature requirements are the same. Symptoms will begin to appear in four to six weeks and within ten weeks will be similar to those shown in Figures 41a and b.
Polyacrylamide gel electrophoresis (PAGE) There is much interest in PAGE techniques for identifying specific bands associated with CEV and CVs. These techniques should be used in conjunction with index plants for identification of specific viroids. There are still many parameters needing research before PAGE technology can fully replace the plant index. It is, however, an excellent tool and should be included in any comprehensive programme for indexing of citrus viroids. Details of the technique are given in Part III.
Nucleic acid hybridization probes
Viroids can be detected by testing for degree of hybridization between sample and labelled probes. Probes may be end-labelled purified viroid or labelled cDNA. Details of the technique are given in Part III.
DETECTION OF EXOCORTIS AND MILD-REACTING CITRON VIROIDS
Summary
Indicators:
861 -S- 1 citron/rough lemon for CEV or CVs.
No. plants/test:
4 to 6 (1 per container) for mild-reacting citrus viroids (CVs),
2 to 3 for strong-reacting CEV
Inoculum:
"Buds" only (no leaf inoculum).
Plant growth:
Single shoot or leader, grown 1 per container.
Temperature:
Warm to hot: 32-40°C max. day/27-30°C min. night.
First symptoms:
CEV - 4 to 10 weeks
CVs - 4 to 6 months
Symptoms:
CEV - Severe leaf epinasty and bark cracking CVs Tip browning
• Petiole wrinkle
• Petiole browning
• Midvein browning
• Mild leaf epinasty
REFERENCES
Benton, R.J., Bowman, F.T., Fraser, L. & Kebby, R.G.1949 Stunting and scaly butt of citrus associated with Poncirus trifoliata rootstock Agr. Caz. NSW, 60: 521-526, 577582 641-645,654
Bitters, W.P., Duran-Vila, N. & Semancik, J.S 1987 Effect of exocortis viroid on flower and fruit structure and development on etrog citron. Plant Disease, 71 397-399
Blue, R.L., Roistacher, C.N., Cartia, G. & Calavan, E.C.1975 Leaf-disc grafting. A rapid indexing method for detection of some citrus viruses In Proc 7th Conf. IOCV, p. 207212 Riverside, IOCV
Calavan, E.C., Frolich, E.F., Carpenter, J.B., Roistacher, C.N. & Christiansen, D.W.1964 Rapid indexing for exocortis of citrus Phytopathol., 54 1359- 1362
Duran-Vila, N., Flores, R. & Semancik, J.S.1986 Characterization of viroid-like RNAs associated with the citrus exocortis syndrome Virol., 150(1): 75-84.
Duran-Vila, N., Pina, J.A., Ballester, J.F., Juarez, J., Roistacher, C.N., Rivera-Bustamente, R. & Semancik, J.S.1988. The citrus exocortis disease: a complex of viroid-RNAs. In Proc. 10th Conf. IOCV, p. 152-164 Riverside, IOCV.
Fawcett, H.S. & Klotz, L.J.1948 Bark shelling of trifoliate orange Calif. Citrogr., 48-230
Fraser, L.R. & Broadbent, P.1979. Virus and virus-related diseases of citrus in New South Wales. Vein enation-woody gall. Dept Agr publication, p 50-51. Chipping Norton, NSW 2170, Australia, Surrey Beatty and Sons
Frolich, E.F., Calavan, E.C., Carpenter, J.B., Christiansen, D.W. & Roistacher, C.N.1965 Differences in response of citron selections to exocortis virus infection In Proc. 3rd Conf. IOCV, p. 113- 118. Gainesville, Univ. Fla Press
Garnsey, S.M.1968. Exocortis virus can be spread by contaminated tools. Citrus Ind., 49: 13-16
Garnsey, S.M. & Jones, J.W.1967 Mechanical transmission of exocortis virus with contaminated budding tools Plant Dis. Rep., 51: 410-413
Garnsey, S.M. & Whidden, R.1973 Efficiency of mechanical inoculation procedures for citrus exocortis virus. Plant Dis. Rep., 57: 886-889.
Nauer, E.M. & Goodale, J.A.1964 Forcing newly budded citrus Calif. Citrogr., 49 294-295, 297.
Nauer, E.M., Holmes, R.C. & Boswell, B.S.1980. Close spacing in the greenhouse inhibits lime seedling growth. HortSci., 15(5): 59 1 -592
Reuther, W., Nauer, E.M. & Roistacher, C.N.1979. Some high temperature effects on citrus growth. J. Am. Soc. Hortis. Sci., 104(3): 353-356.
Roistacher, C.N., Calavan, E.C. & Blue, R.L.1969. Citrus exocortis virus. Chemical inactivation on tools, tolerance to heat and separation of isolates. Plant Dis. Rep., 53: 333336.
Roistacher, C.N., Calavan, E.C., Blue, R.L., Navarro, L. & Gonzales, R.1977. A new more sensitive citron indicator for detection of mild isolates of citrus exocortis viroid (CEV). Plant Dis. Rep., 61: 135-139.
Salibe, A.A. & Moreira, S.1965. New test varieties for exocortis virus. In Proc. 3rd Conf. IOCV. p. 119-123. Gainesville, Univ. Fla. Press.
Schlemmer, A., Roistacher, C.N. & Semancik, J.S.1985. A unique infectious RNA which causes symptoms in citron typical of citrus exocortis disease. Phytopathol., 75: 946-949.
Semancik, J.S.1988. Citrus exocortis disease 1976 to 1986. In Proc . 10th Conf: IOCV, p. 1 36151. Riverside, IOCV.
Semancik, J.S. & Conejero-Tomas, V.1987. Viroid pathogenesis and expression of biological activity. In Viroids and viroid-like pathogens. p. 71 - 126. Boca Raton, Fla., CRC Publishing.
Wallace, J.M.1978. Virus and virus-like diseases. In The citrus industry, Vol. 4. p. 67- 184. Univ. Calif. Div. Agric. Sciences.
Weathers, L.G. 1980. Exocortis. In Bové, J. M. & Vogel, R., eds. Description and illustration of virus and virus-like diseases of citrus. A collection of colour slides. Paris, I.R.F.A. SETCO- FRUITS.
FIGURE 35 Classic severe bark scaling on trifoliate orange rootstock caused by CEV (California)
FIGURE 41 b Severe bark cracking on the stem of citron 861-S-1 indicator plant inoculated with CEV
