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DESCRIPTION AND BACKGROUND
Tristeza is possibly the most destructive disease of citrus. Many millions of trees on sour orange rootstock have been destroyed in Argentina, Brazil, California, Florida and Spain. The disease continues to spread into new areas, e.g. Israel and Venezuela, destroying citrus plantings where sour orange is the predominant rootstock.
There are many strains of tristeza, causing various field symptoms on different scions and rootstocks. Isolates selected from field trees may induce a reaction only in Mexican lime. Some will cause bud-union failure of certain scions on sour orange rootstock, and others can induce severe pitting or stunting and yellows in a variety of indexed seedlings. Tristeza stem pitting can severely injure scions of grapefruit and sweet orange directly, regardless of rootstock, by inducing a severe pitting and enlarged cheesy bark in the scion, resulting in smaller fruit, loss of production and debilitation of the tree. Tristeza stem pitting also seriously affects lime and may limit production in many areas.
Many comprehensive reviews on this disease have been published, i.e. Wallace (1978), Cohen and Bové (1980), Roistacher (1981a,1981b,1982), and Bar-Joseph et al. (1981). Extensive slide and text reviews on many aspects of tristeza are given in Description and illustration of virus and virus-like diseases of citrus, edited by Bové and Vogel (1980), and these are highly recommended for reviewing the tristeza and tristeza seedling-yellows diseases.
The plant index is still invaluable for detection of CTV and its many isolates. Graft inoculation to indicator plants can detect tristeza when virus titre is very low. The severe seedling-yellows and stem-pitting forms of tristeza can currently be distinguished from mild forms only in plants. Garnsey et al. (1987) proposed a standardized host-range analysis for evaluating the severity of tristeza isolates by rating decline, stem pitting and seedling yellows on different hosts.
There are strains of tristeza that are difficult to detect in seedlings of Mexican lime but that can be easily detected by ELISA. For example, a California tristeza isolate (T-519) is very difficult to identify in Mexican lime indicator plants grown under temperature regimes conducive to good symptom development, but it is readily detected by ELISA. This illustrates the value of, and need for, using more than one technique in a programme for the indexing of important foundation block trees.
METHODS OF DETECTION Method 1: field diagnosis
The iodine test. If the rootstock is sour orange and the scion sweet orange, grapefruit or mandarin, a sudden quick decline and wilting, followed by defoliation, especially during the first warm weather of spring, would suggest possible infection by tristeza (Figure 4). A simple field test can be carried out to detect starch depletion in the roots or rootstock below the bud-union. The disappearance of starch from the roots is a result of girdling owing to killing of the phloem cells at the bud-union. Tristeza-induced starch depletion generally proceeds from the outer tips of the roots back toward the trunk of the tree. The application of iodine to the exposed cut surface of a root is a rapid method of testing for starch. The following method is taken from the papers of Bitancourt (1944) and Fawcett (1945):
• Prepare a solution of potassium iodide and iodine by
dissolving 1.5 g of potassium iodide plus 0.3 g of iodine in 100
cc of water. Keep in a coloured glass bottle, away from direct
sunlight.
• Dig up roots (6-mm or 1/4-in diameter or smaller) from the
outer margin of the tree and cut roots at an angle exposing the
inner wood. Place a drop of iodine solution on the cut surface.
Lack of development of a dark blue or black reaction suggests
starch depletion, whereas a strong reaction indicates abundant
starch.
A tree on sour orange rootstock showing quick decline or sudden dieback symptoms and with low starch in the roots would be suspect for tristeza. Verification should be made using ELISA or graft inoculation to Mexican lime seedlings, or both, or any of the diagnostic techniques mentioned in this handbook.
Inverse stem pitting in the sour orange rootstock. A section of bark removed from the bud-union area of a tristeza-infected tree will usually show inverse pitting on the inner surface of the bark with corresponding pegs on the outer surface of the exposed sour orange rootstock (Figure 5). This symptom (on the sour orange rootstock) is highly diagnostic for tristeza.
Stem pitting on scions. Pitting is associated with, and diagnostic for, tristeza. Pits may be seen in the trunk and branches, and vary from severe deep depressions to closely spaced and small (Figures 9b and 13). When pitting is very severe, small branches will snap off readily at the new growth joints. The outer bark may be cheesy and, when peeled, stems may show very small closely spaced or varying sized pits. These can be seen in grapefruit, grapefruit hybrids pummelo, tangelo, limes, various citrus hybrids, and sweet orange, but rarely in lemon, sour orange, trifoliate orange or mandarin. Severe pitting on grapefruit and sweet orange in the field is usually associated with chlorotic, tight, upright branch growth.
Method 2: seedling indexing
Seedling indexing to Mexican lime is still a very powerful tool for detection of tristeza virus. The small-fruited and somewhat seedy Mexican or West Indian lime (Citrus aurantifolia) has various common names such as kaghzi in India, baladi in Egypt, doc in Morocco, and gallego in Brazil. The name key lime is also commonly used. Seedlings of C. excelsa, citron, C. macrophylla or other citrus which show vein clearing and stem pitting can also be used as indicators. However, the Mexican lime is highly sensitive to tristeza and is the preferred indicator.
Collection of budwood. Collect budsticks from a minimum of four quadrants of each tree. For routine reindexing of important foundation or mother-block trees where there may be some danger of possible infection by vector transmission, collect from eight sectors of each tree.
Inoculum tissue. "Buds" (buds with eyes, blind buds or chip buds), leaf discs or leaf pieces can be used (see Part II). Graft two inoculum "buds" or leaf pieces, or a minimum of five or six leaf discs per plant. Since CTV is phloem-limited, it is important that inoculum tissue contain phloem and that cut surfaces of phloem tissue of donor and receptor plants are in good contact.
Inoculation. Place inoculum "buds" or leaf pieces in the lower part of the test seedling, removing as few leaves as possible from the lower stems. The seedling can be cut back to 20-25 cm from the soil surface at the time of inoculation or at two to three weeks after inoculation when wrapping tapes are cut and the inoculum is observed for survival. The time to cut back is decided upon according to the specific environmental conditions in each plant laboratory. With the use of plastic wrapping tapes at the laboratory at Riverside, California, plants are usually cut back at the time of inoculation with very high survival rates of inoculum buds.
Indicator plants. As stated above, Mexican lime is the recommended general indicator for identification of all types of tristeza. To determine if the isolates will cause seedling yellows or stem pitting, inoculate grapefruit seedlings. These are highly sensitive to most CTV isolates, and are the preferred indicator in an initial index for stem pitting (SP-CTV) and/or seedling yellows (SY-CTV). Most seedy grapefruits can be used, and the Duncan variety has been found satisfactory as an indicator. If seedling yellows is found in the grapefruit indicator, then subinoculations can be made from the infected grapefruit to sour orange and sweet orange to determine the severity of the SY-CTV or SP-CTV isolate.
Sour orange is an excellent supplemental indicator for seedling-yellows tristeza and is equally as effective as the Eureka or Lisbon lemon (Figure 1). It is also preferred since it is highly polyembryonic and will produce 70 percent or more plantable seedlings compared with only 8 percent for the highly gametic lemons. Garnsey (unpublished) found that a clonal Eureka lemon grown as a cutting makes an excellent indicator for seedling yellows.
To determine whether an isolate will cause significant stem pitting in sweet orange, it is necessary to inoculate seedlings of a sensitive variety such as Madame Vinous. Although sour orange and lemon seedlings are excellent indicators for seedling yellows, they will rarely show stem-pitting symptoms.
Mexican lime used for general tristeza indexing and grapefruit and sour orange seedlings used for detection of severe CTV isolates can be grown three per container. Using two containers with three plants per container, two plants in each container are inoculated, leaving the third plant as a non-inoculated control. Sweet orange seedlings (Madame Vinous or Pineapple) used for detection of severe CTV-SP isolates should be grown one per container.
Controls. The negative control plant in each container of three is not inoculated. A minimum of one mild- and one severe-reacting tristeza isolate should be included as positive controls in every test. The severe-reacting positive control isolate can be inoculated into two plants in one container and the mild-reacting positive control isolate inoculated into a minimum of four, but preferably six to eight plants. The mildestreacting isolate available should be used, i.e. one that induces very few leaf or stem-pitting symptoms in Mexican lime (Figures 6b and 9a).
Positive controls for seedling yellows or stem pitting should be "buds" taken from a minimum of two reactive sources with known symptomatology and should be inoculated into two grapefruit and/or sour orange seedlings per container. A strong and a mild reactive isolate are preferred. In large-scale tests for seedling yellows, two or more known reactive isolates should be used. Negative controls must always be included.
Inoculum survival. The wrapping tapes should be cut and removed two to three weeks after inoculation and the survival of the graft inoculum recorded. Although tristeza is not readily transmitted mechanically, other pathogens are, thus it should be standard procedure to dip all tools prior to cutting into any plant (in a 1 percent sodium hypochlorite disinfectant solution).
Leaf-disc grafts can be evaluated one to two weeks after inoculation. If one of the two "bud" or leaf-piece grafts is alive, the plant need not be reinoculated. However, if both bud and leaf-piece grafts are dead, or if three or more of the five to six leaf-disc grafts are dead, then plants should be reinoculated or new plants inoculated.
Post-inoculation plant care. The developing young side shoots on the Mexican lime seedlings should not be trimmed for the first three flushes of growth, or for approximately eight weeks, in order to obtain the maximum number of leaves to examine for vein clearing. Occasionally only a bottom shoot will have leaves with symptoms. However, after the third growth flush, the side shoots should be trimmed and the most vigorous terminal shoot tied to a stake and trained to grow as a single shoot for later examination for stem pitting (Cachexia Figure 48). If not trimmed, Mexican lime seedlings have a tendency to send out many lateral shoots and, when grown three to a container, can quickly overcrowd the container and bench. Single shoots permit more light, thicker stem growth and better pesticide spray coverage. Trimmed plants are also easier to handle, and a single vigorous shoot produces a large single stem that can be readily peeled for critical observation of stem pitting.
Grapefruit, sour orange and sweet orange index seedlings used for seedling-yellows index tests should be trained as a single leader or shoot, starting with the first dominant emerging shoot. Seedlings of these varieties have a natural tendency to develop as single shoots, and little trimming is needed. Supplemental lighting during the winter will significantly aid the growth of lime and sour orange seedlings but may not benefit grapefruit seedling growth much.
Temperature requirements. Cool temperatures are necessary for maximum tristeza symptom expression in plants. Warm temperatures above 35°C may suppress development of vein-clearing and stem-pitting symptoms in Mexican lime seedlings (Roistacher et al.,1974). The preferred greenhouse temperatures for all tristeza and seedling-yellows indexing are 24-28°C maximum during the day and 17-21 °C minimum at night. Temperature control is especially important when checking for mild isolates.
Time of symptom development. Figure 2 shows the time in weeks for the first appearance of vein-clearing symptoms in the leaves of 355 tristeza-inoculated Mexican lime seedlings. Plants were inoculated with a number of tristeza isolates over a seven-year period at Riverside, California. Within the first five weeks, 88 percent of inoculated plants developed leaf symptoms, and within eight weeks 97 percent of plants were diagnosed as positive. Figure 3 shows the time for the first appearance of seedling-yellows symptoms in grapefruit, sour orange and lemon seedlings. Over 90 percent of seedlings developed seedling-yellows symptoms within nine weeks, and some showed symptoms after as little as five weeks. This is based on observation of 1200 positive control plants inoculated with seven selected severe isolates over a five-year period.
Symptoms of tristeza
Vein clearing. The primary symptom in both the young and mature leaves of the Mexican lime is intermittent translucent vein clearing (Figures 6a, 6b and 7). The symptoms are best observed if the leaf is held overhead so that sunlight shines directly through the brightly exposed leaf. For critical reading of mild leaf-flecking symptoms, plants may have to be taken out into direct sunlight if the greenhouse is shaded.
Vein-clearing symptoms in leaves of Mexican limes can be readily detected in plants inoculated with most CTV isolates. However, vein-clearing symptoms induced by some mild-reacting isolates may be difficult to see. Only a few leaves may show an occasional mild fleck in the vein (Figure 6b). A non-inoculated control seedling in each container is most helpful in judging the vein-clearing symptoms in the leaves of inoculated plants. Mild vein-clearing symptoms do not usually persist in mature leaves. Plants should be observed frequently when new flushes are developing. The optimum period for observation is just as a leaf becomes fully expanded.
If the underside of leaves of lime or sweet orange is observed by reflected light, the veins will usually show distinct dark greenish-black, "water-soaked" areas (Figure 6c). These same areas, when viewed through sunlight, will show strong vein clearing. These "water-soaked" areas may persist in mature leaves after vein clearing is masked.
Leaf cupping Figure 7 shows typical leaf cupping associated with tristeza in Mexican lime. Leaf-cupping symptoms are usually pronounced when plants are grown at cool temperatures and under good growth conditions, but they may not always be present. However, leaf cupping is also a symptom induced in leaves of Mexican lime seedlings by the vein-enation virus in the complete absence of tristeza and, therefore, in the absence of other symptoms, leaf cupping alone may not be diagnostic for tristeza. Leaf cupping usually remains after the leaf matures or hardens. It may be pronounced when plants are infected with severe CTV isolates.
Vein corking. Very severe isolates of seedling yellows tristeza may induce a corking on the veins of Mexican limes, sweet orange or grapefruit very similar to symptoms induced by boron deficiency. The vein corking can be mild, or severe as shown in Figure 8.
Stem pitting. For most CTV isolates, pitting can be observed after about eight weeks by peeling back the bark and observing the peeled stem. However, pitting is best evaluated at the end of the index test about four to six months after inoculation when the bark of the large single shoots can be completely peeled and the stems carefully observed. If the bark does not peel readily, steaming the stems in an autoclave is helpful for loosening tight bark.
Most tristeza isolates, except perhaps the very mildest, induce pitting in Mexican lime seedlings. Pits may be few (Figure 9a) or numerous (Figure 9b). The pitting symptom in the stems of Mexican lime or any other seedling indicator is highly diagnostic for tristeza. Observations of many thousands of tristeza-infected Mexican lime seedlings have indicated that stem pitting is strongly associated with vein clearing in the leaves.
Observation of stem pitting is very useful to confirm diagnosis when conditions are not optimum for leaf symptoms.
Symptoms of seedling-yellows tristeza. When three plants of grapefruit, sour orange or sour lemon are grown in an individual container and two are infected with seedling yellows, the symptoms are clear and dramatic as shown in Figures 10 and 11. Stunting may be severe, moderate or mild. The leaves are usually smaller, chlorotic, sometimes yellow, and may have pointed tips (Figure 12), and the shoots are compressed, thus giving the plant a stunted appearance.
Symptoms of stem-pitting tristeza. Figures 13a to 13d show the stem-pitting reaction in stems of grapefruit, sour orange, sweet orange and rough lemon. respectively.
Almost any seedling of any variety can be pitted by some specific tristeza isolate. Most CTV isolates, however, do not cause pitting in mandarin, sour orange, sour lemon, rough lemon or sweet orange seedlings or trees. Stem pitting can be very severe in limes, Citrus macrophylla, grapefruit, grapefruit hybrids, tangelos and certain pummelo cultivars, and may limit their use where severe isolates are endemic. Certain CTV isolates may severely pit sweet orange. The Pera orange of Brazil is particularly susceptible. and a severe stem-pitting isolate affects navel orange trees in Peru.
When grapefruit seedlings infected with seedling-yellows tristeza show severe stunting, as in Figure 10, pitting may be difficult to evaluate since infected seedlings are too small and stems too thin. Inoculations into larger seedlings may be necessary to force vigorous shoots if pitting is to be seen and judged. Some isolates of CTV will induce pitting without inducing stunting or yellows reaction in grapefruit or sweet orange (Roistacher, Dodds and Bash,1988).
Occasionally severe CTV isolates may induce a thick "cheesy" ,bark, usually associated with many very tine pits. This symptom is highly diagnostic both in field trees and in inoculated plants.
Termination
Tristeza reaction in Mexican an lime. When shoots reach about 1 m (approximately four to six months from inoculation), one or two of the mild-inoculated positive control plants should be harvested, the bark peeled and the peeled stems critically observed for pits. If pitting is evident in the control plants which had been inoculated with the very mildest-positive isolates, and if vein-clearing symptoms were observed and recorded in past readings in these mild-positive controls, then all plants can be harvested, peeled and examined for pits. Pitting can he recorded as none, mild, moderate or severe on a scale of 0 to 3 or 0 to 5. If no pitting is found in the mild-positive controls, the plants should be cut back, new shoots forced and the evaluation procedure repeated.
If no pits are seen in the mild-positive control plants, ELISA should be used to verify presence or absence of CTV. A modified procedure has been incorporated into the Citrus Clonal Protection Program at Riverside, California, which combines both the plant index and ELISA. "Buds" from budsticks collected from foundation block trees are first inoculated into Mexican lime seedlings.
After about eight weeks at optimum temperatures, the lime plants are trimmed to single shoots, with the exception of a side shoot which is allowed to develop. When this side shoot reaches a length of about 20 to 25 cm, it is harvested, and the bark peeled and processed for ELISA. In this manner, the tissue used for the ELISA has been grown under optimum temperature conditions in the greenhouse, and the low titre problem associated with seasonal temperature variation (Dodds et al.,1987) is circumvented. This combined procedure of plant index and ELISA uses the best of both index tests to assure freedom from tristeza in prime budwood source plants.
The T-519 tristeza isolate, which showed little or no vein clearing or stem pitting, has consistently indexed strongly positive by ELISA.
Seedling yellows and stem pitting. When three plants are grown per container, most symptoms of seedling yellows can be seen fairly dramatically within ten weeks after inoculation, as indicated in Figure 3. However, the seven isolates used in this study represent selected severe-reacting seedling-yellows. Milder-reacting isolates would take somewhat longer to induce reactions. Also, as mentioned before, some stem-pitting isolates may show no yellows reaction in grapefruit or sweet orange but may show severe pitting. Therefore, all grapefruit or sweet orange plants should be maintained until they reach about 1 m in height before harvest. When possible. known positive stem-pitting controls should be used.
The sour orange used for seedling-yellows indexing rarely shows stem pitting and need not be peeled in routine indexing. The index test can be terminated when the milder-reacting seedling yellows positive controls show definitive symptoms. This may not be until three to four months after inoculation.
Method 3: ELISA
The use of ELISA for detection and diagnosis of tristeza is now a well-tested and proven technique and should be incorporated into any indexing programme.
It is an excellent technique for surveys and large-scale testing, for obtaining very rapid results and for verifying the presence or absence of CTV isolates that are mild reacting in indicator plants. However, at present it cannot be used to distinguish between various isolates of tristeza, nor should it be relied upon as the sole index for testing Important foundation or primary budwood sources. Practical details and procedures are set out in Part III.
New methods of identifying CTV strains or isolates by use of differential hybridization techniques (Rosner, Lee and Bar-Joseph,1986) may, in the future, provide rapid identification of certain seedling-yellows and stem-pitting isolates, which currently can be distinguished only by long-term plant index.
Method 4: microscopic detection of inclusion bodies
Inclusion bodies of CTV may be seen in sectioned tissue by light microscopy. This can permit a very rapid means of tristeza identification.
Details for sectioning fresh or fixed citrus tissue for detection of CTV are given in Part III, and have been reviewed by Garnsey et al. (1980).
Miscellaneous
CTV detection by dsRNA analysis. A specific band in stained electrophoretic gel is diagnostic for tristeza (Dodds, Tamaki and Roistacher,1983) and there is evidence that certain seedlingyellows isolates are distinguished from tristeza isolates by specific band migration locations. Details are given in Part III.
Detection of CTV by electron microscopy. CTV virus particles can be detected and identified within minutes by dicing small quantities of tissue in a specific buffer, placing it on grids and observing it in an electron microscope. This is the most rapid method of detecting and diagnosing CTV (Garnsey et al., 1980).
TRISTEZA DETECTION Summary
Graft transmission to Mexican lime
Indicator:
Mexican lime.
No. plants/test:
3 (plus 1 control in each of 2 containers).
Inoculum:
"Buds", leaf pieces, or leaf discs.
Plant growth:
Allow all shoots to develop for the first three growth flushes,
then prune and train as a single shoot.
Temperature:
Cool: 24-27°C max. day/18-21°C min. night.
First symptoms:
3 to 5 weeks (end of first or second growth flush).
Symptoms:
Vein clearing, vein darkening, leaf cupping, and stem pitting.
Vein corking for severe isolates.
SEEDLING YELLOWS DETECTION
Summary
Graft transmission to seedling indicators
Indicators:
Seedlings of grapefruit, sour orange.
No. plants/test:
2 (plus 1 control in each of 2 containers).
Temperature:
Cool: 24-27°C max. day/18-21°C min. night.
Plant growth:
Train as a single shoot.
First symptoms:
Within 10 weeks.
Symptoms:
Small yellow leaves, compact growth, stunted plants. Distinctly
smaller and more compact than control.
STEM-PlTTING DETECTION
Summary
Indicators:
Seedlings of grapefruit, and Madame Vinous or Pera sweet orange.
No. plants/test:
2 to 4 (grown 1 per container).
Temperature:
Cool: 24-27°C max. day/18-21°C min. night.
Plant growth:
Train as a single shoot.
First symptoms:
Within 4 months.
Symptoms:
Pits in grapefruit stems can be very severe. Less frequent in
sweet orange stems, but pitting can be very severe with certain
isolates. Thick, cheesy bark evident with some isolates in
grapefruit. Seedlings of grapefruit or sweet orange may or may
not show seedling yellows.
REFERENCES
Bar-Joseph, M. et al.1981. A review on the epidemiology of the tristeza disease, an ongoing threat.4th International Citrus Congress, Tokyo (Japan), Nov.1981. In Proc. Int. Soc. Citriculture, 1: 419-423.
Bitancourt, A.A.1944. Um teste pare a identificação precoce da tristeza dos citrus. O Biológico, 10(6): 169-175.
Bové, J.M. & Vogel, R., eds.1980. Description and illustration of virus and virus-like diseases of citrus. A collection of colour slides. Paris, I.R.F.A. SETCO-FRUITS.
Cohen, M. & Bové, J.M.1980. Tristeza. In J.M. Bové & R. Vogel, eds. Description and illustration of virus and virus-line diseases of citrus. A collection of colour slides. Paris, I.R.F.A. SETCO-FRUITS.
Dodds, J.A., Tamaki, S.J. & Roistacher, C.N.1983. Indexing of citrus tristeza virus double stranded RNA in field trees. In Proc. 9th Conf. IOCV, p. 327329. Riverside, IOCV.
Dodds, J.A. et al.1987. Effect of strain, host, time of harvest and virus concentration on doublestranded RNA analysis of citrus tristeza virus. Phytopathol., 77(3): 442-447.
Fawcett, H.S.1945. A starch test for quick decline. Calif. Citrogr., 30: 122.
Garnsey, S.M. et al.1980. Detection of citrus tristeza virus. II. Light and electron microscopy of inclusions and viral particles. In Proc. 8th Conf. IOCV, p. 916. Riverside, IOCV.
Garnsey, S.M. et al.1987. Towards a standardized evaluation of the biological properties of citrus tristeza virus. Phytophylactica,19: 151 - 157.
Roistacher, C.N.1981 a. A blueprint for disaster. Part 1: The history of seedling-yellows disease. Citrograph, 67: 4-5,24.
Roistacher, C.N.1981b. A blueprint for disaster. Part 2: Changes in transmissibility of seedling yellows. Citrograph, 67: 28-32.
Roistacher, C.N.1982. A blueprint for disaster. Part 3: The destructive potential for seedling yellows. Citrograph, 67: 48-53.
Roistacher, C.N., Dodds, J.A. & Bash, J.A.1988. Cross-protection against citrus tristeza seedling yellows (CTV-SY) and stem pitting (CTV-SP) viruses by protective isolates developed in greenhouse plants. In Proc. 10th Conf. IOCV, p. 91-100. Riverside, IOCV.
Roistacher, C.N. et al.1974. Suppression of tristeza virus symptoms in Mexican lime seedlings grown at warm temperatures. Plant Dis. Rep., 58(8): 757-760.
Rosner, A., Lee, R.F. & Bar-Joseph, M.1986. Differential hybridization with cloned cDNA sequences for detecting a specific isolate of citrus tristeza virus. Phytopathol., 76(8): 820824.
Wallace, J.M.1978. Virus and virus-like diseases. In The citrus industry, Vol.4, p.167- 184. Univ. Calif. Div. Agric. Sciences.
FIGURE 7 Cupping on leaves of Mexican limes inoculated with tristeza virus. Note also vein clearing
FIGURE 12 Close-up of seedling-yellows reaction
