Psorosis complex: psorosis-A, psorosis-B and ringspot
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DESCRIPTION AND BACKGROUND
The psorosis disease of citrus was first observed in Florida and California in the early 1890s and named psorosis based on the Greek psora = ulcer or mange. The disease was commonly called scaly bark. It originated in the Orient and was spread worldwide by the distribution of citrus species and varieties. Fawcett (1938) first proved transmissibility by graft-inoculation with buds from infected to non-infected trees.
Fawcett (1933) designated the disease as psorosis-A to distinguish it from a more virulent form called psorosis-B. Psorosis-A has been linked with the concave gum-blind pocket and crinkly-leaf infectious variegation diseases of citrus as one complex (Fawcett and Bitancourt,1943). However, recent evidence suggests they are all separate diseases, and their distinct differences in symptomatology and cross protection are discussed in detail in the respective sections. The psorosis disease was comprehensively reviewed by Timmer and Beñatena (1977) and reviewed and illustrated by Roistacher (1980). Typical psorosis-A trunk lesions on a field tree are shown in Figures 72a and 72b, and internal wood staining in the cross-section of a branch is seen in Figure 72c.
The agent responsible for the psorosis disease of citrus is almost certainly a virus, though it has not as yet been purified or characterized. Derrick et al.. (1988) reported that two components are associated with the citrus ringspot virus. These components can be isolated, and when mixed together are infectious. Cross-protection of the milder forms of psorosis-A against a challenge with the severe psorosis-B is diagnostic for classifying a disease as belonging to the psorosis-A complex.
The major susceptible varieties showing bark scaling are sweet orange, mandarin and grapefruit. The sour orange, sour lemon and rough lemon show no external bark symptoms. Most citrus species and cultivars are symptomless carriers of the virus. Citrus diseases that show bark lesions and also somewhat resemble psorosis-A are Rio Grande gummosis of grapefruit and leprosis of sweet orange. Indexing of budwood from suspect trees by graft transmission to specific indicator seedlings should readily distinguish these diseases from psorosis-A.
The psorosis complex of viruses has many variants and contains isolates ranging from those which are non-mechanically transmissible to some which are, such as ringspot or the very serious and spreading Argentinian psorosis. Twenty-one isolates of psorosis-A, obtained from various field trees at the University of California citrus variety collection at Riverside, were graft-inoculated into a variety of indicator plants and tested for mechanical transmissibility from citron to citron. There was much variability in symptoms found in citron, Dweet tangor and sweet orange among the various isolates, and only two isolates transmitted mechanically. However, despite this variability, all 21 isolates in sweet orange rejected a challenge from psorosis-B, confirming their identity as psorosis-A.
The disease is spread primarily by humans via propagation of infected budwood. It has been observed that 50-year-old sweet orange tree showing no bark lesions in the field produced progeny trees with over 60 percent psorosis bark lesions (Roistacher and Calavan, unpublished). Thus, although psorosis may remain symptomless in certain host trees, the virus can be transmitted from a symptomless host and induce symptoms in progeny trees. Mechanical, seed or root-graft transmission occurs with various psorosis types. Ringspot will move by mechanical transmission in the field (Timmer and Garnsey,1980). Currently a severe form of psorosis is spreading fairly rapidly in Argentina and is relatively destructive (Figure 72b). The means of spread has not been determined, but vector transmission is suspected.
The first use of citrus seedlings to detect a graft-transmissible pathogen in citrus was by Wallace (1945) for detection of psorosis-A. This seedling index reduced the time required for indexing from an average of approximately 11 years, for development of bark lesions in field trees, to about six weeks for symptom development in the young leaves of sweet orange. This was a revolutionary development that pioneered this relatively rapid detection of citrus pathogens by indexing to greenhouse-grown plants.
There has been much confusion about the relationship between concave gum and psorosis diseases since both may cause leaf fleck in field trees or indicator plants. Roistacher and Calavan (1965) separate these two diseases for the following reasons:
Psorosis-A causes a distinct bark scaling in sweet
orange, mandarin or grapefruit (Figures 72a and 72b), whereas
concave gum causes concavities in sweet orange or mandarin trunks
and branches (Concave gum, Figure 81) and does not induce
scaling. These are separate and distinct symptoms.
A tree infected with the concave gum pathogen will usually show a series of concentric gum rings in a cross-section of the trunk or branch (Figure 82; also Cristacortis, Figure 93a), whereas trees infected with psorosis-A usually show a specific wood staining in similar sections of the trunk or branch (Figure 72c). These symptoms are very distinct.
Many leaves on trees infected with the concave gum pathogen will show diagnostically strong oak-leaf patterns (OLP) (Figure 83; also Cristacortis, Figure 95 and Impietratura, Figure 89) in the developing leaves during the spring growth flush under cool conditions, whereas leaves on trees infected only with the psorosis-A pathogen rarely show symptoms in the spring growth flush except if co-infected with the concave gum pathogen. Leaves of trees infected with some of the more severe forms of psorosis, i.e. ringspot, may show strong patterns distinctly different from OLP (Figures 73a, 74 and 75b) in the young and mature leaves in the field, but rarely show typical OLP in the spring flush unless mixtures of pathogens are present.
When inoculated into seedlings of sweet orange or mandarin, the concave gum pathogen induces a specific mild flecking in leaves of the early flushes, and the oak-leaf pattern usually develops in leaves of later flushes. Shock symptoms are rare. Inoculations with psorosis-A infected tissue will rarely show OLP unless in a mixture, and will usually show shock symptoms in the young flush of seedling growth under cool temperature conditions.
Concave gum-infected sweet orange plants will not protect against a challenge from psorosis-A or psorosis-B infected tissue.
METHODS OF DETECTION Method 1: Field diagnosis
Psorosis-A can be diagnosed in the field if the two symptoms of bark scaling and wood staining of stems are observed (Figure 72). Bark scaling alone, though usually diagnostic, should not be totally relied upon for identification. Psorosis-like bark lesions, which apparently are not associated with a graft-transmissible pathogen, have been observed in several areas. The psorosis-B and ringspot forms of psorosis may show varying fruit symptoms (as shown in Figure 73) or ringspot leaf patterns (as shown in Figures 73a and 74). These symptoms are highly diagnostic for the ringspot form of psorosis. Field trees showing bark lesion symptoms of psorosis-A in California, South Africa, the Mediterranean region, Brazil and occasionally elsewhere usually do not show leaf patterns on the young growth flushes on mature trees (Doidge, 1939; Passos, 1965; Roistacher, 1980). Thus, field observation for leaf symptoms is not recommended for diagnosis of psorosis-A.
Method 2: Seedling index
Indexing to seedlings is the principal method for the positive diagnosis for the psorosis-A complex.
Budwood collection. A minimum of four budsticks are collected from each quadrant of a candidate or test tree. If a tree in the field is selected as a prime candidate for thermotherapy or shoot-tip grafting, a budstick should be taken in the proximity of a typical fruit, and a bud propagation made from this budstick held in the greenhouse or plant laboratory. This greenhouse propagation becomes the primary plant, and budwood can be taken anywhere from this plant for indexing or for use as positive control tissue for testing the effectiveness of thermotherapy or shoot-tip grafting.
Indicator plants. The sweet orange seedling is the preferred indicator, and Pineapple, Madame Vinous or Olivelands sweet orange have been found to be superior varieties for detection of psorosis-A (Roistacher and Nauer,1964). There are definitive varietal differences in sensitivity to psorosis-A and such varieties as Koethen, Mediterranean or Diller sweet oranges should not be used (Roistacher and Nauer,1964). Mandarins are acceptable indicators but do not always show the shock symptoms associated with psorosis-A, and they may not be so sensitive to lesion formation in cross-protection tests. Although citron and lemon seedlings are excellent indicators for psorosis, they are sensitive to exocortis, other pathogens and tristeza, and psorosis symptoms may be masked or confused in the presence of these pathogens. If seedling-yellows tristeza is not endemic, the grapefruit seedling makes an excellent indicator for certain ringspot isolates.
At least four seedlings (two each in two containers) should be inoculated per test. These should be grown three seedlings per container; two are inoculated and one left as a non-inoculated negative control in each of the two containers.
Inoculum tissue and inoculation. A minimum of two inoculum "buds" (buds, blind buds or chip buds) per seedling are recommended. They are placed anywhere in the lower part of the seedling, leaving as many leaves in place as possible. The seedling can be cut off at about 2025 cm from the soil surface at the time of inoculation, or two to three weeks after inoculation when tapes are cut and bud survival rate is recorded.
Controls. As indicated above, a non-inoculated negative control should be included in each container. Also, if available, at least two psorosis isolates should be included as positive controls in each test: one mild and the other a known standard that consistently induces shock symptoms in inoculated seedlings.
Inoculum survival. Two to three weeks after inoculation, the wrapping tape should be cut and removed and the inoculum "buds" examined for survival. Although most psorosis isolates are not mechanically transmissible, some are, and mechanical transmission can be prevented by disinfecting the cutting tool by dipping it into a 1 percent sodium hypochlorite solution when going from plant to plant. Any dead or dying inoculum "buds" should be recorded, and if both "buds" are dead the plant should be reinoculated or a new plant used.
Post-inoculation care and temperatures. The temperature during the first four weeks after inoculation is critical for symptom expression. Cool temperatures will favour the appearance of shock reactions in the young emerging shoots (Figure 75), whereas warm temperatures may inhibit shock reactions and mask leaf symptoms. Psorosis-A symptoms are best expressed at relatively cool temperatures of 24-27°C maximum day and 18-21 °C minimum night. Shock and leaf-pattern symptoms may not appear if temperatures are too warm (Figure 75).
After the sweet orange seedling is cut back, the new growth should not be trimmed or suckered but permitted to grow or flush freely. The critical period for development of shock and young leaf symptoms is during the first and second flushes of growth.
If possible, the use of insecticide spray should be avoided during this critical period of symptom development.
Supplemental light may enhance symptom development and should be used during the winter months (see section on supplemental lighting in Part II).
Symptoms and time for development of first symptoms. Shock symptoms may show as soon as the first shoots emerge (after three or four weeks). Symptoms of shock are first observed in young emerging shoots as a wilt-like drooping (Figure 76a) followed by the drying up of these young "shocked" shoots (Figure 76b). The leafless shoot is twisted, turns brown and necrotic and ultimately withers and dries up. It is later seen as a dried-up remnant which remains with the plant permanently (Figures 75a and 76b). Shock can be complete, i.e. all emerging shoots dry up and lose their leaves. This is especially evident when plants are inoculated with severe isolates of psorosis-A or ringspot.
More typically, shock may occur only on a few emerging shoots. The shoots that do not shock will usually show varied types of young leaf symptoms, as seen in Figure 77. These appear four to six weeks after inoculation. As the leaves mature, the symptoms may disappear from the hardened leaves. However, some isolates of psorosis and those of ringspot will show symptoms which persist in mature leaves, as illustrated in Figures 73a, 74, 75b and 78. Plants which lose all their first flush of leaves because of complete shock will usually show young leaf symptoms in the next flush of growth. The new emerging shoots of the second growth flush usually do not show shock symptoms.
Shock symptoms may not occur if temperatures are too warm. Also the intensity of symptoms will vary depending on temperature and/or the isolate. Leaf symptoms may not occur or may be different (Figure 75b) if greenhouse temperatures are too warm.
Most of the leaf symptoms will appear in the first two flushes of growth. Later flushes may or may not be symptomatic depending upon the isolate and temperature. It is advisable to observe the developing leaves of the inoculated plants critically during the first two flushes of growth or approximately during the first eight weeks.
Termination. The presence of shock symptoms and young leaf symptoms in the positive controls. observed within the first eight weeks after inoculation, is sufficient to make a judgement of presence or absence of psorosis-A. If the object is only to ascertain the presence or absence of the psorosis pathogen, this eight-week period is sufficient, provided that the positive control plants inoculated with the non-heat-treated or non-shoot-tip grafted bud source are positive. If the object is to identify as fully as possible the reaction observed in the sweet orange test plants, then the three sweet orange seedlings growing in each container (two inoculated and one non-inoculated control) should be permitted to grow and be trained as single shoots. These inoculated plants will then be challenged with psorosis-B as given in Method 3.
Method 3: Cross-protection
To determine if the virus is psorosis-A, an inoculated sweet orange seedling is challenge inoculated with psorosis-B lesion inoculum and observed for evidence of cross-protection.
Challenging with inoculum of psorosis-B. A source of psorosis-B is needed for use in cross protection tests. This is obtained by grafting lesion inoculum, taken from trunk-bark lesions of a field tree, to a sweet orange seedling or budling (see procedure for bark grafting in Part II). In six to eight weeks, under proper temperature conditions, blister-like lesions should form on the stems (Figure 79a), and later develop on the leaves (Figure 79b). The twig lesion inoculum is then used to challenge the sweet orange index plants which had shown leaf patterns, and to determine whether they belong to the psorosis-A family.
Psorosis-B source plants should be maintained in a cool greenhouse or screenhouse and should preferably show the bark-blister symptoms shown in Figure 79a.
Challenge inoculation. Two "buds" from blister inoculum, as shown in Figure 79a, are graft inoculated into one of the two pre-inoculated sweet orange indicator plants to be challenged, originating from Method 2. The non-inoculated control seedling is similarly inoculated, and the third plant is held as the pre-inoculated control. Plants can be cut back or left. Within eight to 12 weeks blister-like lesions should form on the challenged, non-protected control seedlings. The psorosis-B lesions usually form near the initial challenge inoculation site.
If lesions develop on the pre-inoculated and challenged test plants, then psorosis-A is not indicated. Conversely, if lesions do not develop on the pre-inoculated challenged test plants, but develop abundantly on the non-pre-inoculated challenged controls, then the virus in question is most probably related to the psorosis-A virus complex.
Method 4: Mechanical transmission of certain psorosis isolates and citrus ringspot virus
Citrus ringspot virus has been mechanically transmitted from citrus to citrus and from citrus to herbaceous hosts. The method given below for mechanical transmission from citrus to herbaceous hosts is based on Garnsey and Timmer (1980).
Inoculum tissue and buffer. The best tissue is from young leaves of recently inoculated citrus plants in the shock phase. Leaves are ground in a pre-chilled mortar and pestle with cold 0.05 M TME tris buffer at pH 8.0, plus 0.5 percent (V/V) 2-mercaptoethanol.
Inoculation. Chenopodium quinoa is grown from seed in a well-fertilized and aerated soil mix to produce vigorous succulent growth. Artificial supplemental lighting should be used during the winter months. Almost fully expanded leaves of C. quinoa are dusted with 500-mesh carborundum and the inoculum applied with a cotton swab.
Symptoms. Symptoms will appear in four to six days as chlorotic local lesions distributed over the leaves, as illustrated in Figure 80.
Garnsey and Timmer (1980) succeeded in mechanically transmitting Florida, Texas and California ringspot isolates, plus three California psorosis-B isolates from citrus to Chenopodium quinoa. They could not mechanically transmit any isolate showing symptoms in C. quinoa back to citrus from C. quinoa, but could transmit ringspot isolates from C. quinoa to Gomphrena globosa and then back to citrus from G. globosa.
Mechanical transmission from citrus to citrus is best carried out by knife or razor slash into the stem. Citron is an excellent host and receptor plant. A slicing cut is first made into the branch of symptomatic citron tissue and then into the stem of the receptor citron. Ten slices can be used as a standard. The sliced stem is then wrapped securely with budding tape.
Many psorosis-A isolates will not transmit mechanically from infected sweet orange or citron to other citrus or herbaceous hosts (Roistacher, unpublished). Therefore, a negative response to mechanical inoculations does not indicate freedom from virus, and does not eliminate the need for bud transmission to citrus indicator plants.
Madame Vinous or Pineapple sweet orange seedlings.
No. of plants/test:
4 seedlings (3 plus 1 control in each of 2 containers).
"Buds" (buds, blind buds or chip buds).
Allow all shoots to develop after initial cut-back.
Cool: 24-27°C max. day/18-21°C min. night.
Shock, followed by young leaf flecking and mottle in leaves of new shoots.
Chenopodium quinoa .
No. of plants/test:
8 (3 plus 1 control in each of 2 containers).
Young symptomatic citrus leaf tissue - leaves or leaf tips (preferably tissue in shock).
0.05 M TME tris buffer pH 8.0; leaf rubbed with carborundum.
2 1 -27°C.
4 to 6 days.
Chlorotic local lesions, well distributed.
Derrick, K.S. et al.1988. Two components associated with the citrus ringspot virus. In Proc.10 th Conf. IOCV, p. 340-342. Riverside, IOCV.
Doidge, E.M.1939. Scaly bark or psorosis of citrus trees in South Africa. Dept. of Agric. and Forestry Science Bull., 208 pp.
Fawcett, H.S.1933. New symptoms of psorosis indicating a virus disease of citrus. Phytopathol., 23: 930. (Abstract)
Fawcett, H.S.1938. Transmission of psorosis of citrus. Phytopathol., 28:669. (Abstract)
Fawcett, H.S. & Bitancourt, A.A.1943. Comparative symptomatology of psorosis varieties on citrus in California. Phytopathol., 33: 837-884.
Garnsey, S.M. & Timmer, L.W.1980. Mechanical transmission of citrus ringspot virus isolate from Florida, Texas and California. In Proc. 8th Conf: IOCV, p. 174-179. Riverside, IOCV.
Passos, O.S.1965. Absence of young leaf symptoms of psorosis in the State of Bahia, Brazil. In Proc . 3rd Conf: IOCV, p. 167- 169. Gainesville, Univ. Fla. Press.
Roistacher, C.N.1980. Psorosis-A. 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.
Roistacher, C.N. & Calavan, E.C.1965. Crossprotection studies with strains of concave gum and psorosis viruses. In Proc . 3rd Conf. IOCV, p. 154-161. Gainesville, Univ. Fla. Press.
Roistacher, C.N. & Nauer, E.M.1964. A comparison of certain sweet orange varieties as indicators for concave gum and psorosis viruses. Plant Dis. Rep., 48: 56-59.
Timmer, L.W. & Benatena, H.N.1977. Comparison of psorosis and other viruses causing leaf flecking in citrus. In Proc . Int. Soc . Citriculture, 3: 930-935.
Timmer, L.W. & Garnsey, S.M.1980. Natural spread of citrus ringspot virus in Texas and its association with psorosis-like diseases in Florida and Texas. In Proc. 8th Conf. IOCV, p. 167- 173. Riverside, IOCV.
Wallace, J.M.1945. Technique for hastening foliage symptoms of psorosis of citrus. Phytopathol., 35: 535-541.
FIGURE 72a Scaly bark symptoms of psorosis-A on the trunk of a sweet orange in Calitornia
FIGURE 72b Severe scaly bark lesions of psorosis on a nucellar sweet orange tree in Concordia, Argentina
FIGURE 72c Staining of interior wood of a branch of sweet orange infected with psorosis-A. This symptom is diagnostic for psorosis-A when found on trees showing bark lesions as in (a)
FIGURE 73a Symptoms of ringspot psorosis on leaves and fruit of Navelina orange in Greece showing indented sunken areas on fruit
FIGURE 73b Symptoms of ringspot on fruit of Clementine mandarin in Spain showing green spotting of fruit. Impietratura may be present
FIGURE 74 Leaf symptoms induced by ringspot psorosis inoculated to seedlings of Marsh grapefruit (California)
FIGURE 75a The effect of temperature on symptom expression, showing shock symptoms of psorosis-A on Inoculated sweet orange seedlings grown under relatively cool temperatures of 24-27°C maximum day (left) compared with no symptoms on plants grown under relatively warm temperatures of 32-385C maximum day (right) (California)
FIGURE 75b The effect of temperature on symptom expression, showing variation In feat symptoms due to the effect of the above temperature regimes. The leaf on the left is from a plant grown under the cool temperature and the feat on the right is from a plant grown under the warm temperature regime. Both plants were inoculated with the same inoculum
FIGURE 76A Psorosis-A shock symptoms in inoculated seedlings of Madame Vinous sweet orange showing the wilt-like drooping of the new shoot. The young leaves will dry up and drop off
FIGURE 76b Psorosis shock symptoms in Inoculated young seedlings of Pineapple sweet orange. The plant on the left is completely shocked. Shock remnants will remain on the plant and do not drop off
FIGURE 77a Young feat symptoms In Dweet tangor. A non-inoculated control feat Is on the right
FIGURE 77b Young leaf symptoms In Pineapple sweet orange. Mild psorosis-A symptoms In leaves of Plneapple sweet orange with non-inoculated control leaf on the right
FIGURE 78a Mature ringspot type leaf symptoms in mature leaves of Valencia orange (Photo: L.J. Klotz)
FIGURE 78b Mature ringspot type leaf symptoms in mature leaves of rough lemon
FIGURE 79a Lesion bark blisters of psorosis-B on a twig and thorn of sweet orange (above) with the noninoculated control below
FIGURE 79b Psorosis-B-induced, blister-like lesions on the underside of a sweet orange leaf. Similar lesions may be found on leaves of field trees infected with ringspot
FIGURE 80 Local lesions on leaves of Chenopodium quinoa induced by mechanical inoculation from ringspot or psorosis-Binfected citrus tissue (control leaves on right)
DESCRIPTION AND BACKGROUND
Concave gum and blind pocket diseases of citrus were first observed and named by a grower, J.C. Perry, in Highland, California, in the early 1930s and shown to H.S. Fawcett, who described the disease (Fawcett,1936). Concave gum and blind pocket are probably part of the same complex with blind pocket showing sharper, deeper depressed concavities. Roistacher and Calavan (1965) separated concave gum from psorosis-A based on: leaf symptoms on field trees; leaf symptoms on inoculated indicator seedlings; trunk symptoms; internal wood staining or gumming; and cross-protection reactions. These differences are reviewed in the psorosis-A section.
The relationships of the various leaf-flecking-type diseases are discussed in detail in a review by Timmer and Beñatena (1977). Concave gum and other diseases which induce oak-leaf pattern (OLP) symptoms are found mostly in the Mediterranean countries but can also be found less frequently in most of the citrus-growing areas of the world. Concave gum is a disease that primarily induces symptoms in sweet orange, mandarins, tangors and tangelos, with many citrus cultivars retaining the pathogen as symptomless hosts. Although the pathogen has not been isolated, it is almost certainly a virus and has shown cross-protection by strains that induce mild OLP symptoms against a challenge with severe OLP-inducing strains (Roistacher and Calavan,1965). It is readily transmitted by grafting and topworking, usually by people, but can be naturally root-grafted to adjacent trees. There is a possibility that certain related isolates may be seed-transmitted (Bridges, Youtsey and Nixon,1965). Pollen from concave gum-infected trees, when placed under the bark of indicator plants, will transmit the disease (Vogel and Bové,1980). Concave gum disease has not been shown to be vector or mechanically transmissible. The pathogen can be readily eliminated from infected budwood by thermotherapy or by shoot-tip grafting in vitro.
METHODS OF DETECTION Method 1: Field diagnosis
Concave gum disease can be diagnosed in the field by three types of symptoms. The first is the presence of distinct concavities in the trunks of sweet oranges, mandarins or tangelos, as illustrated in Figure 81. For other descriptions, slides and photographs of field symptoms see Klotz (1976) and Wallace (1978). A second diagnostic indication of concave gum disease is the presence of concentric gum rings in cross sections of large twigs and branches as shown in Figure 83 and Cristacortis, Figure 93a. This symptom is usually found in association with concavities in the trunk. Vogel (personal communication) reports that certain varieties such as Washington navel and Orlando tangelo will show good symptoms of gum rings in the wood, whereas certain sweet oranges such as Tarocco, or some mandarins such as Willow leaf, do not.
A third diagnostic field symptom, usually found in association with the first two, is the presence of OLP in nearly all of the young developing leaves in the spring flush of growth and also in other flushes developing under cool temperature conditions (Figure 83; Cristacortis, Figure 95 and Impietratura, Figure 89).
The OLP symptom is also found in association with impietratura and cristacortis diseases. When OLP is found in leaves of field trees, although indicating the presence of a disease, it is not specific for any of the three mentioned diseases. In warm or hot climates, the OLP symptom may not be apparent if the leaf flushes develop when temperatures are too warm. However, Garnsey (personal communication) reports that concavities can occur on Minneola tangelo in Florida in the absence of OLP symptoms. Certain mandarin trees will naturally develop concavities that are not associated with infection but are natural to the variety. Indexing is indicated.
Method 2: Seedling index
The seedling index used for detection of interveinal clearing and OLP typically associated with concave gum disease is the same for the diagnosis of all three of the OLP diseases, i.e. concave gum, cristacortis and impietratura. The general seedling index procedures for all three are as follows:
Collection of budwood. At least four budsticks should be collected, one from each quadrant of a candidate tree. If a tree in the field is selected as a prime candidate whose budwood will be heat-treated or shoot-tip grafted, a budstick should be taken below or proximal to a well developed and typical fruit (see Part II).
Inoculum tissue. "Buds", i.e. buds with "eyes", blind buds or chip buds, are preferred as inoculum tissue for grafting, although other tissue can be used. A minimum of two inoculum "buds" per seedling is recommended.
Inoculation. Inoculum "buds" are placed anywhere in the lower part of the seedling, leaving as many leaves in place as possible. The seedling can be topped or cut at about 20-25 cm from the soil surface at the time of inoculation, or two to three weeks after inoculation when tapes are cut and bud survival is recorded.
Indicator plants. The Dweet tangor seedling has been found to be a most sensitive indicator variety for expressing the OLP symptom. However seedlings of certain mandarins, i.e. Dancy, Kara or King, are also excellent indicators (Roistacher,1963).The sweet orange seedling is not as effective as Dweet tangor or certain mandarins in detecting the mild isolates of OLP inducing pathogens, and some of these mild reacting isolates can be missed entirely in leaves of most sweet orange seedlings (Roistacher and Nauer,1964; Roistacher and Calavan,1965). However, sweet orange is still a very effective indicator for expressing OLP for most isolates of the concave gum pathogen.
At least four plants, two each in two containers, should be inoculated per test. These should be grown as three seedlings per container; two are inoculated and one left as the non-inoculated control.
Controls. In each container of three seedlings, one should be left as a non-inoculated control. A severe- and a mild-positive control (if available) should be included in each test and inoculated respectively into a minimum of two plants in the three-plant container, with the third plant as the non-inoculated negative control.
Inoculum survival. Two to three weeks after inoculation, the wrapping tape should be removed to expose the inoculum for examination. Although concave gum has not been found to be mechanically transmissible, it is good sanitary procedure in a plant laboratory to disinfect any tool used for cutting into a plant by dipping it into a 1 percent sodium hypochlorite solution when going from plant to plant. Any dead or dying inoculum "buds" should be recorded and, if both inoculum "buds" are found dead, the plant should be reinoculated using fresh inoculum if available or the original budsticks stored in the refrigerator.
Post-inoculation care. The new growth of the Dweet tangor or mandarin indicator seedlings should not be trimmed but allowed to flush freely. The most critical period for observing OLP is usually at the second flush of growth after inoculation, and the young leaves should be inspected frequently for symptoms during this flush period. Insecticide sprays should be avoided if possible during this critical flushing period. Interveinal flecking and OLP symptoms appear in the expanding leaves of the young developing flush, and may disappear completely as the leaves mature and harden. The milder flecking symptoms usually disappear in the hardened mature leaves.
Supplemental lighting, when applied during the winter months, will enhance OLP symptom expression. For example, at Riverside, California, at 34°N latitude, the addition of five hours of 40- to 50-foot candles of supplemental light (at the plant level) during the months of October to April induced symptoms in 207 leaves on plants of four indicator varieties compared with only 60 leaves on plants grown without light (Roistacher,1963). In addition to the more than threefold increase in the number of leaves showing symptoms under artificial lights, there was a 32 percent increase in the total number of leaves produced (see Supplemental lighting in Part II).
Temperature requirements. The leaf patterns associated with concave gum and related diseases are best seen in inoculated indicator plants grown under cool temperatures similar to those for tristeza, psorosis etc. (24-30°C maximum day and 18-21°C minimum night). Under warm conditions, symptoms may not appear. Cool night temperatures and moderate day temperatures characteristic of spring are best for maximum symptom expression. Since symptoms of OLP are best seen in the leaves of growth flushes during the cooler spring or autumn periods, conditions in the plant laboratory should simulate the naturally occurring temperatures as much as possible.
Time for development of symptoms. The first symptoms will appear on emerging leaves within five to eight weeks, usually with the second flush of growth.
Symptoms. The first symptom is usually mild interveinal leaf flecking similar to that in Figure 83b. Whereas psorosis-A-type leaf flecks develop in the very early young leaves, concave gum symptoms of interveinal clearing and OLP are best observed in the more developed young leaves. OLP will most frequently develop in the larger leaves, and is best seen before the leaf hardens (Figure 83). The oak-leaf pattern may appear as a thin, translucent delineation on each side of the midvein (as in Figure 83b) or as the fully expanded, translucent imprint of an oak leaf (Figure 83a; Cristacortis, Figure 95 or Impietratura, Figure 89).
Termination. The index test can be concluded when leaves of both the mild and severe positive control plants show clear definitive symptoms in all of their respective inoculated plants. Experience at the Rubidoux laboratory in Riverside, California, has shown that with the proper indicator plants, which have been grown under optimum nutrition, temperature and supplemental light conditions, positive controls will usually show symptoms within an eight week period.
If space is not a critical factor, test plants can be held for one additional flush after controls have shown symptoms.
CONCAVE GUM (OAK-LEAF PATTERN) DETECTION
Seedlings of Dweet tangor, King, Kara or Dancy mandarin, or sweet orange.
4 plants (2 plus 1 control in each of 2 containers).
"Buds" (buds, blind buds, chip buds).
After inoculation and cut-back, allow seedlings to develop full flushes. Do not trim.
Cool: 24-27°C max. day/18-21°C min. night.
5 to 8 weeks; best in second or third flush.
Interveinal flecking followed by oak-leaf patterns in emerging young leaves. Symptoms may fade in mature, hardened leaves.
Bridges, G.D., Youtsey, C.O. & Nixon, R.R.1965. Observation indicating psorosis transmission by seed of Carrizo citrange. In Proc. Fla. State Hort. Soc., 78: 48-50
Fawcett, H.S.1936. Citrus diseases and their control. New York, McGraw-Hill. 656 pp.
Fawcett, H.S. & Bitancourt, A.A.1943. Comparative symptomatology of psorosis varieties on citrus in California. Phytopathol., 33: 837-884
Klotz, L.J.1976. Concave gum. In Bové, J.M. & Vogel, R., eds. Description and illustration of virus and virus-like diseases of citrus. A collection of colour slides and text. Paris, l.R.F.A. SETCO-FRUITS.
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FIGURE 81 Concavities associated with concave gum diseases in the trunk of a sweet orange tree in the field (Spain)
FIGURE 82a Concentric gum rings in a cross-section of a branch. This is highly symptomatic for concave gum disease when present with trunk concavities and OLP (Photo: R. Vogel)
FIGURE 82b Concentric gum rings in a cross-section of a small twig. This is also symptomatic for concave gum disease (Photo: R. Vogel)
FIGURE 83a Strong oak-leat patterns In the leaves from a field tree of Valencia orange showing concavities during the spring flush of growth
FIGURE 83b A narrow oak-leaf pattern developed about the midvein In a leaf of Valencia orange. Note the strong interveinal flecking, usually the first symptom observed in leaves on plants inoculated with concave gum virus
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