Tatterleaf

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DESCRIPTION AND BACKGROUND

The tatterleaf disease of citrus, induced by the citrus tatterleaf virus (CTLV), was first described by Wallace and Drake (1962) as a transmissible disease that induced mottled and tattered leaves in citrus excelsa indicator seedlings. Calavan, Christiansen and Roistacher (1963) first showed the destructive potential of this disease to citrange rootstock when tatterleaf-infected tissue was graft-inoculated to satsuma mandarin budded on Troyer citrange rootstock (Figure 56). Meyer (Beijing) lemon trees, which were first imported into the United States from Beijing (China) in 1908, were later found to contain the tatterleaf virus. Many Meyer lemon trees worldwide that originated from the 1908 introduction probably contain the virus, including many propagations and plantings of the original Beijing lemon in China (Zhang, Liang and Roistacher,1988). The disease is endemic in mainland China and may be widespread (Zhang et al.,1988). Tatterleaf disease is widespread in Taiwan Province and in Japan and is probably present elsewhere where Meyer lemon or other infected citrus have been imported from these countries. It has been reported from South Africa in declining Shamouti orange trees on citrumelo rootstock.

Most citrus species and all commercial cultivars are symptomless carriers of the virus. Symptoms will appear as bud-union crease (Figures 55 and 56), or as a fluting and reduction of the stock as in Figure 56, where infected scions are grafted to trifoliate orange or its hybrids. When the bud-union crease is severe, the tops may shear off at the union in high winds (Figure 57). Miyakawa and Tsuji (1988) report that some isolates of CTLV do not cause bud union crease. Trifoliate orange is immune to tatterleaf and the virus is unequally distributed in citrange.

Semancik and Weathers (1965) showed mechanical transmission of CTLV from citrus to cowpea and partially purified the virus. It was rod-shaped, 19 by 650 nm, and transmissible to 19 herbaceous hosts. Wallace and Drake (1968) suggested that two viruses were present (tatterleaf and citrange stunt) since shoots of inoculated C. excelsa indicator seedlings would recover after showing symptoms, and these recovered shoots contained a transmissible agent which would react in citrange but not in C. excelsa. Recovered c excelsa shoots could be reinfected by the virus present in Meyer lemon buds and showed tatterleaf symptoms. They called the new virus "citrange stunt". Roistacher (1981,1988) showed that recovered shoots of C. excelsa graft inoculated to symptomless carriers would eventually show the tatterleaf component. He suggested that the disease was one complex and the original name "tatterleaf' be retained to describe both diseases.

CTLV is difficult to eliminate from budwood by shoot-tip grafting but was eliminated from budwood by thermotherapy (Roistacher,1977). Koizumi (1984) eliminated the virus from citrus tissue by combining shoot-tip grafting with thermotherapy. Recently, Navarro and coworkers (unpublished) succeeded in eliminating CTLV from citrus tissue by shoot-tip grafting in vitro.

The virus is readily mechanically transmitted from infected citron to citron by knife or razor cuts, and the virus can be inactivated on tools by dipping them in a 1 percent sodium hypochlorite solution. CTLV was noted as spreading from tree to tree at the South Coast Field Station in southern California, presumably by mechanical transmission (Roistacher, unpublished). It is important that indexing for CTLV should be included in any programme for establishing primary foundation trees since many citrus species and commercial cultivars are symptomless carriers and the virus is highly mechanically transmissible. CTLV can be very destructive to citrus on trifoliate rootstock or their hybrids (Figures 55-57).

METHODS OF DETECTION

Method 1: Index to indicator plants

Collection of budwood. Collect a minimum of four budsticks from each quadrant of the tree to be indexed.

Inoculum tissue. "Buds" (buds, blind buds or chip buds).

Indicator plants. Seedlings of Rusk, Troyer or Carrizo citrange, citremon and C. excelsa are used. If tristeza is endemic, omit the C. excelsa because it is very sensitive to citrus tristeza virus, and symptoms of tatterleaf may be readily masked. Mexican lime in the absence of tristeza may show pronounced psorosis-like symptoms. Rusk citrange is more sensitive than Troyer or Carrizo citrange (Miyakawa,1980). Zhang et al. (1988) reported that Troyer citrange was superior to Carrizo citrange as an indicator. Indicators can be used as seedlings, as a clonal budline topworked or budded to a plant containing the virus, or budded as a scion on to any inoculated seedling and forced. Grow indicator plants preferably three per container and inoculate at least four plants, two in each container, leaving the third plant as a negative control. Where Rusk or other citrange buds are to be forced as scions, grow one rootstock seedling per container.(See method described for forcing scions under "Cachexia".)

Inoculation. Use at least two "buds" to inoculate each plant. Cut back seedlings at about 20-25 cm above the soil surface. The seedling may be cut back at the time of inoculation, or two to three weeks later when inoculum survival is recorded. If citrange is used as a forced scion, the rootstock seedling should preferably be bent just above the scion bud (Cachexia Figure 47) or cut back or topped just above the scion bud.

Controls. Both positive and negative controls should be included. Where seedlings are used and grown three per container, one of the three should be left as a negative control for each index test. Positive CTLV control buds should be inoculated into two of the three plants in one container, leaving the third plant as a negative control.

Inoculum survival. Cut the grafting tapes two to three weeks after inoculation and record "bud" survival. Reinoculate only if both inoculum "buds" are dead. Since CTLV is highly mechanically transmissible on tools, dip the razor- or knife-blade used for cutting the grafting tape in a 1 percent solution of sodium hypochlorite between plants.

Post-inoculation care. After the initial cut-back, allow seedlings or the budded Rusk citrange scions to develop all shoots without trimming. Supplemental light will enhance growth of citrange during winter months and should be incorporated in the plant laboratory as normal procedure (see Part II).

Temperature requirements. Tatterleaf is a cool temperature virus and warm temperatures have been observed to mask symptom development.

Maintain index room temperatures at 24-30C maximum day (or preferably cooler), and 1821 C minimum night.

Time for first symptoms. Five to seven weeks in C. excelsa and six to eight weeks or longer in Rusk or other citrange. Occasionally, delayed reaction may result in the appearance of symptoms beyond eight weeks.

Symptoms. The first symptom is a mild chlorotic spotting of the leaves, usually only in the first flush of growth. Later growth will show intense leaf spotting, leaf deformation and stunting. The leaves look "tattered" as if the edges were torn in a non-uniform pattern (Figures 58 and 59). Symptoms in citrange are clear spots that persist and develop into chlorotic spots. Stems are blotched. Symptoms persist in the mature leaves. Inoculated plants of C. excelsa may develop "recovered" or symptomless shoots after the first or second flush of growth. These are the symptomless shoots that will induce a reaction in citrange or citremon but not in C. excelsa (the citrange stunt component of the tatterleaf complex) (Wallace and Drake,1968).

Termination. Allow two to three growth flushes to develop (eight to 12 weeks), or wait until the positive control plants show strong and well-developed symptoms.

Method 2: Mechanical transmission to herbaceous hosts

The method recommended for mechanical transmission is based on the procedure of Garnsey (1974), which has consistently produced reliable index results. However, it must be remembered that sap inoculation techniques require higher concentrations of virus in the inoculum than is necessary for graft-transmission, and sensitivity is sacrificed for speed of assay.

Inoculation procedure. Young, succulent leaf tissue is macerated in cold neutral 0.05M potassium phosphate buffer at a ratio of 0.1 g of tissue to 1 ml of buffer. Plants are dusted with 500-mesh carborundum and leaves are inoculated with a cotton swab or fingers immersed in inoculum. Leaves are rinsed with tap water after inoculation and the plants are incubated in moderate light at 21-24C.

Indicator plants. The cowpea Vigna unguiculata subsp. unguiculata (syn. Vigna sinensis) variety Early Ramshorn, other cowpea cultivars and the common red kidney bean can be used. Chenopodium quinoa has been used successfully in Taiwan Province. Grow five plants per container and inoculate four plants in each of two containers, with the fifth plant as the non-inoculated control. Bean and cowpea plants are usually ready to inoculate eight to 12 days after seed planting and under good growing conditions.

Symptoms. Symptoms will develop in four to six days. Symptoms in cowpea and red kidney bean are chocolate-brown necrotic lesions in the primary leaves (Figure 60). Mosaic patterns may appear in the secondary trifoliate leaves of cowpea under certain conditions. Symptoms in C. quinoa are chlorotic local lesions on inoculated leaves and a systemic mottle or mosaic (Figure 61). Citrus ringspot produces chlorotic local lesions in C. quinoa and is also systemic. Local lesions are seen in red kidney bean in Figure 62.

MISCELLANEOUS

Attempts are now in progress to develop good antisera to CTLV. If these become available, ELISA will be a viable option for indexing.

TATTERLEAF DETECTION

Summary
• Graft transmission to citrus

Indicators:
Citrus excelsa and/or Rusk or other citrange or citremon as seedlings. Rusk, or other citrange buds forced on any rootstock except trifoliate orange.

No. plants/test:
8 seedlings (3 plus 1 control in each of 2 containers). Grow 1 per container for forced scion buds.

Inoculum:
"Buds" (buds, blind buds or chip buds).

Plant growth:
Allow all shoots to develop.

Temperature:
Cool: 24-27C max. day/18-21C min. night.

First symptoms:
5 to 7 weeks.

Symptoms:
Chlorotic mottle, leaf spotting with deformed leaves. C. excelsa leaves look tattered. Plants are severely stunted. Stem distortion and blotching in citrange.

• Transmission to herbaceous hosts

Indicators:
Cowpea, red kidney (Phaseolus vulgaris) bean, Chenopodium quinoa.

No. plants/test:
8 plants (4 plus 1 control in each of 2 containers).

Inoculum:
Young succulent leaf tissue in 0.05 to 0.1M phosphate buffer, pH 7.0.

Inoculation:
Rub carborundum-dusted leaves with inoculum.

Temperature:
Cool (21-24C).

First symptoms:
4 to 6 days.

Symptoms:
Chocolate-brown necrotic lesions in primary leaves of cowpea and red kidney bean; mosaic patterns may appear in secondary leaves of cowpea. Chlorotic local lesions in inoculated leaves of C. quinoa. A systemic mottle or mosaic in secondary growth of C. quinoa with leaf distortion.

REFERENCES

Calavan, E.C.,Christiansen, D.W. & Roistacher, C.N.1963. Symptoms associated with tatterleaf virus infection of Troyer citrange rootstock. Plant Dis. Rep., 47: 971 -975.

Garnsey, S.M.1974. Mechanical transmission of a virus that produces tatterleaf symptoms in Citrus excelsa. In Proc. 6th Conf. IOCV,p. 137-140. Gainesville, Univ. Fla. Press.

Koizumi, M.1984. Elimination of tatterleaf-citrange stunt virus from satsuma mandarin by shoot-tip grafting following pre-heat treatment. In Proc . 9th Conf. IOCV, p. 229-233. Riverside, IOCV.

Miyakawa, T.1980. Occurrence and varietal distribution of tatterleaf-citrange stunt virus and its effect on Japanese citrus. In Proc. 8th Conf: IOCV, p. 220-224. Riverside, IOCV.

Miyakawa, T. & Tsuji, M.1988. The relationship of tatterleaf reaction to bud-union crease of Poncirus-rooted citrus trees. In Proc . 10th Conf: IOCV. Riverside, IOCV.

Roistacher, C.N.1977. Elimination of citrus pathogens in propagative budwood. I. Budwood selection, indexing and thermotherapy. In Proc . Int. Soc. Citriculture, 3: 965-972.

Roistacher, C.N.1981. Attempts to separate components of the tatterleaf citrange stunt complex. In Proc. Int. Soe. Citriculture, 1: 430433.

Roistacher, C.N.1988. Citrus tatterleaf virus: further evidence for a single virus complex. In Proc.10th Conf: IOCV, p. 353-359. Riverside, IOCV

Semancik, J.S. & Weathers, L.G.1965. Partial purification of a mechanically transmissible virus associated with tatterleaf of citrus. Phytopathol.,55: 1354-1358.

Wallace, J.M. & Drake, R.J.1962. Tatterleaf, a previously undescribed virus effect on citrus. Plant Dis. Rep., 46: 211 -212.

Wallace, J.M. & Drake, R.J.1968. Citrange stunt and ringspot, two previously undescribed virus diseases of citrus. In Proc. 4th Conf. IOCV, p. 177- 180. Gainesville, Univ. Fla. Press.

Zhang, T.M., Liang, X.Y. & Roistacher, C.N.1988. Occurrence and detection of citrus tatterleaf virus (CTLV) in Huangyan, Zhejiang Province, China. Plant Diseuse, 72: 543-545.

FIGURE 55 Bud-union crease of satsuma mandarin on trifoliate rootstock induced by citrus tatterleat virus (CTLV). Specimen at Kuchinotsu, Japan

FIGURE 56 Severe tatterleaf reaction induced by CTLV showing bud-union crease and fluting in the trunk of Troyer citrange rootstock with satsuma orange scion (Photo: E.C. Calavan)

FIGURE 57 Breakage and separation of a tatterleat-infected satsuma scion from its trifoliate rootstock. Specimen at Kuchinotsu, Japan

FIGURE 58 Symptoms of tatterleaf in leaves of Rusk citrange inoculated with CTLV. Note deformed leaves with spots and blotches

FIGURE 59 Symptoms of tatterleaf in leaves of Citrus excelsa from a seedling inoculated with CTLV. The noninoculated control leaf is on the left.. Note the tattered, irregular-edged leaves giving the disease its name

FIGURE 60 Symptoms of necrotic local lesions in the primary leaves of cowpea induced by mechanical transmission with CTLV from citrus tissue

FIGURE 61 Symptoms in secondary growth leaves of Chenopodium quinoa showing well-distributed spotting, chlorosis and distortion induced by mechanical leaf-rub inoculation with CTLV

FIGURE 62 Local lesions in a leaf of red kidney bean mechanically inoculated with CTLV (Photo: S.M. Garnsey)

Infectious variegation and leaf rugose

DESCRIPTION AND BACKGROUND

The citrus infectious variegation virus (CIVV), also called citrus variegation virus (CVV), was the first citrus virus transmitted experimentally. Trabut (1913) named it "infectious chlorosis" and transmitted it to other citrus. Fawcett and Klotz (1939) described it as a new disease called "infectious variegation". They found symptomatic lemon trees in Glendora, California, and transmitted the disease to sour orange. A similar disease found in Florida by Grant and Smith (1960) was transmitted to many other citrus species. Fawcett (1936) placed this disease in the psorosis family.

CIVV is of exceptional interest since it was the first citrus virus to be mechanically transmitted from citrus to citrus and to herbaceous hosts. In addition to its presence in the United States, the disease has been reported from many countries in the Mediterranean basin and from Australia.

Crinkly leaf, once thought to be a separate virus, was shown to be a milder strain of CIVV (Majorana and Martelli,1968). CIVV will be used to designate both mild and severe strains. CIVV is serologically related to other ilarviruses, i.e. asparagus viruses II-P and II-S, citrus leaf rugose virus, elm mottle virus and Tulare apple mosaic virus (Uyeda and Mink,1983). It is fairly similar to the satsuma dwarf family of viruses in its morphology, particle size and reaction on certain citrus seedling indicator plants. However, there are differences in components, no evidence for serological relationship and distinct differences in mechanical transmission to herbaceous hosts.

Citrus leaf rugose virus (CLRV) was described by Garnsey (1975) as a citrus crinkly-leaf type, and found in one location in Florida. It has subsequently been found in Japan and Argentina. It induces flecking in Eureka lemon, leaf rugosity in Mexican lime and severe stunting of young Duncan grapefruit plants. It is an ilarvirus, 25-32 nm in diameter. Two strains, which differ in their effect on Duncan grapefruit, have been described. It appears to be similar to the infectious variegation group but with some distinct serological differences (Garnsey,1975; Garnsey end Gonsalves,1976).

A variegation which occurs on leaves of sour orange called "Petri's variegation" was reported from Italy (Petri,1931). Leaf symptoms are identical to those of CIVV, but Petri's variegation is non-transmissible and is probably a result of genetic sensitivity of the local sour orange seedling to winter cold (Majorana and Scaramuzzi,1965).

Although both Fawcett (1936) and Wallace (1957) placed crinkly-leaf and infectious variegation in the psorosis family as it is based primarily on leaf symptoms, they should be considered as separate and distinct citrus virus diseases. There are no serological relationships; CIVV will not protect plants against a challenge from psorosis-A or psorosis-B; symptomatology on leaves (specifically the leaf curl is distinct), and CIVV does not cause the scaling reaction in trunks of sweet orange, grapefruit or mandarin scions induced by psorosis-A. CIVV can be separated from mixtures with psorosis-A or other psorosis-like pathogens by mechanical transmission from citrus to citrus or from citrus to herbaceous hosts. For a complete description, slides and report on CIVV, see Desjardins and Bov (1980) and Wallace (1978).

Mechanical transmission of the virus from citrus to citrus by tools can be prevented by dipping tools in a 1 percent solution of sodium hypochlorite. CIVV can be transmitted at low percentages through seed (Wallace,1968); vector transmission has not been reported. Transmission of CIVV by humans via infected budwood or by mechanical means is the primary means of spread. CIVV can be eliminated from citrus tissue by thermotherapy and by shoot-tip grafting in vitro. The purification and production of antiserum have been done for some strains of CIVV, and rapid identification by immunodiffusion techniques (Garnsey,1975) and by ELISA (Davino and Garnsey,1984) has been reported.

METHODS OF DETECTION Method 1: Field diagnosis

The primary symptoms of CIVV in field trees of lemon, sweet orange, mandarin or grapefruit are seen in the leaves, which show distorted, puffed or puckered leaf segments with or without variegation (Figure 63). When present, this puckered leaf symptom is strongly suggestive of the presence of CIVV. Field symptoms of CLRV are generally inconspicuous.

Garnsey et al. (1984) showed that mild strains of CIVV may exist which induce no symptoms on leaves of field trees. Therefore indexing is imperative for any budwood selection being considered for a mother or foundation tree. Also, leaf symptoms of Petri's variegation appear identical to those caused by CIVV on leaves of sour orange and perhaps other cultivars and, again, indexing is imperative for diagnosis. At times aphid damage may induce symptoms on leaves of field trees suggesting presence of CIVV. Indexing would readily differentiate aphid damage from virus infection.

Method 2: Indexing by graft transmission to indicator seedlings

Collection of budwood. Collect budsticks from twigs showing leaves with distinct symptoms. If no symptoms are evident in the field tree, collect four budsticks per tree, one from each quadrant.

Inoculum tissue. "Buds" (buds, blind buds or chip buds).

Inoculation. Graft a minimum of two "buds" to each indicator seedling. Seedlings can be cut back at the time of inoculation, or two to three weeks after inoculation when wrapping tapes are cut or removed and inoculum survival is recorded.

Indicator plants. Citron or lemon seedlings are the preferred indicators, and should be used for critical evaluation of mild strains. Lemon should be used for citrus leaf rugose virus. If exocortis is present, lemon or other indicators can be used to avoid interference. Dweet tangor, mandarin, sour orange or sweet orange seedlings are other options. Use two containers with three plants per container; inoculate two plants in each container, leaving the third as a non-inoculated control.

Controls. In addition to the negative controls, a mild- and a severe-positive control should be included for each index test. A very mild Florida source, e.g. CVV-2 if available, would make an excellent mild-positive control.

Inoculum survival. After two to three weeks, cut the wrapping tapes securing the inoculum "buds" and record bud survival. It is important that the knife or razor-blade used to cut the tapes be ,US Horticulture Research Laboratory, ARS, USDA, 2120 Camden Road, Orlando, FL 32803, United States of America dipped in a 1 percent sodium hypochlorite solution between plants.

Post-inoculation care. After the initial cut-back at time of inoculation, allow the new growth of all emerging shoots to develop without trimming. Ensure that plants are maintained free of insect pests. To prevent leaf damage from insecticidal spray or residue, use a water spray for pest control and, if possible, withhold insecticide spray treatment until after the first or second flush of growth. (See Part II for insect control in the greenhouse.)

Temperature requirements. CIVV and CLRV are cool-temperature viruses and indexing should be done at 24-30C daytime maximum and 18-21C night-time minimum.

Time for appearance of first symptoms. Symptoms for most isolates of the CIVV family should appear in the first flush of growth within four to six weeks after the initial cut-back, or more definitely in the second flush of growth six to eight weeks after the initial cut-back. Symptoms of mild isolates may take longer to appear.

Symptoms. Symptoms induced in citron by severe CIVV isolates are extreme leaf distortion, epinasty and a pronounced chlorotic mottle (Figure 64). The presence of exocortis viroid may cause confusion or distortion and epinasty symptoms in citron; therefore other indicators should be used. Leaves of lemon, sweet orange and sour orange will show similar leaf patterns in the early young flushes. When the flush hardens, typical puckered elevated segments may remain (Figure 65). These symptoms are similar to the field symptoms shown in Figure 63. Dweet tangor or rough lemon seedlings make excellent indicators for observation of young leaf symptoms which are similar to those induced by the psorosis-A pathogen (Figure 66).

When mild isolates such as the Florida CVV-2 are inoculated into citrons, leaves will show a mild mottle (Figure 67), some epinasty, and perhaps some mild shock in the first growth flush. Subsequent growth flushes may show a transitory mild mottle in the younger leaves which disappears in the mature leaves. An occasional leaf may show the puckered or elevated leaf symptom. Leaf flecking in lemon is the most consistent symptom for this mild strain.

Symptoms of CLRV are: variable rugosity of leaves of Mexican lime, pinpoint chlorotic flecks on expanding leaves of Eureka lemon without leaf distortion, and severe stunting and chlorosis on seedlings of Duncan grapefruit.

Termination. If the mild-positive control Florida CVV-2 is used, the index can be terminated when definitive positive symptoms are observed in citron or lemon, as in Figure 67. In the absence of controls, hold indicator plants for three to four months under cool, spring-like temperature conditions. If new growth leaves are free of symptoms and the severe-positive controls have all shown strong symptoms, the test can be terminated.

For critical indexing of candidate trees for inclusion in a foundation block, it is desirable to inoculate herbaceous hosts and include ELISA in the indexing procedures.

Method 3: Inoculation to herbaceous hosts

The mild transient symptoms induced in citrus indicator plants by the Florida isolate CVV-2 suggest that similar mild isolates could easily be missed in an indexing programme based only on graft inoculation to citrus indicator plants. Procedures are available for reliable testing for CIVV to herbaceous hosts.

A number of herbaceous plants have been reported as showing positive reaction when mechanically inoculated with CIVV-infected citrus tissue. Three indicators have been selected for their ability to detect the published strains of CIVV, i.e. the California, Florida and Italian isolates, plus the Florida leaf rugose and CCV-2 isolates.

Inoculum tissue. Young leaf tissue or young growing tips can be collected directly from the field tree or greenhouse propagation, put in an ice chest and taken to the laboratory for inoculation. The young leaf tissue from the field tree or from the greenhouse propagations is macerated with a chilled pestle and mortar in sufficient 0.05 M neutral potassium phosphate buffer to create a 1:10 dilution. Aknown positive source of CIVV should be included in each index test, plus non-inoculated negative controls. If the very mild positive Florida isolate CVV-2 can be obtained, it should be included as a mild positive control (see footnote under Method 2).

Indicators. The three indicators suggested are: Vigna unguiculata subsp. unguiculata (syn. Vigna sinen.sis), Early Ramshorn or California No. 5 cowpea; Phaseolus vulgaris, Bountiful or red kidney bean; and Crotalaria .spectabilis. Five seeds are sown or transplanted; four seedlings are inoculated, and one left as a non inoculated control. A minimum of four inoculated plants per container is recommended per index variety, and preferably eight inoculated plants in two containers should be used.

Inoculation. Leaves of the indicator plants are dusted with 500-mesh carborundum and the sap inoculum applied with a cottonwool swab. Inoculation must be done as rapidly as possible after maceration. Plants of cowpea and bean are rub-inoculated on their primary leaves before the character leaves appear. The Crotalaria is inoculated at the four- to eight-leaf stage. Plants should be rinsed with water immediately following inoculation.

Controls. Positive and negative controls must always be included.

Post-inoculation care. Temperatures should be relatively cool. A temperature range of 18-24C is satisfactory and 20-22C is best. Some shading after inoculation may be beneficial; symptoms in C. spectabilis increase with intensified sunlight. Light conditions will affect the type of local lesion produced in cowpea, which can vary from a diffuse chlorotic lesion to a well-defined necrotic lesion. Local lesions produced by CLRV in bean are small and necrotic. Holding plants under partial shading after inoculation and then transferring them to normal light should induce good symptoms in the three test indicator species.

Time for first symptoms. Local lesions may begin to show on cowpea or red kidney bean in two to five days with CLRV. However, CIVV will show local lesions on cowpea but not on bean. Necrotic ringspot lesions will begin to appear in four to five days in leaves of C. spectabilis. The brilliant chlorotic vein banding in bean leaves occurs within seven to 14 days. However, the time for appearance of symptoms will be somewhat variable depending upon temperature, light, indicator variety and severity of the isolate.

Symptoms in cowpea. Various symptoms will occur depending on strain, light and temperature conditions. With Florida CIVV, chlorotic/necrotic lesions occur in the primary (inoculated) leaves and systemic mottle may or may not occur in the secondary growth leaves. Two types of local lesions occur on the primary leaves: some are reddish and necrotic (Figure 68). and some are chlorotic spots. Approximately 25 percent of plants may show systemic symptoms in ten to 30 days. Symptoms are yellow chlorosis of the veins, mosaic mottle, vein necrosis and leaf curl (Figure 69).

Symptoms in red kidney bean. Small necrotic local lesions may appear in primary leaves infected with leaf rugose virus. The systemic reaction in secondary growth leaves for all CIVV isolates is usually apparent within seven to 14 days and shows brilliant vein clearing and pronounced yellow vein banding (Figure 70). Some isolates are milder than others.

Symptoms in C. spectabilis. Brownish necrotic ringspot lesions appear early in the primary leaves. The spots may enlarge slightly, and vein necrosis occurs when spots come in contact with veins. Symptoms range from necrotic fleck with chlorotic halo to a necrosis of the terminal portion in eight to 12 days or longer depending on temperature, light and isolate (Figure 71).

Method 4: ELISA

ELISA has been used to index for CIVV from both field and greenhouse sources (Garnsey et al.,1984: Davino and Garnsey,1984). Young spring flush growth is the best source for field collected samples. Antisera to mild and severe isolates of CIVV (CVV) react equally well to all isolates tested.

Antisera to CLRV are also available and react very well in ELISA tests. The serological cross reaction between CLRV and CIVV (CVV) is not very strong with the polyclonal antisera available. A weak positive test may be obtained to CLRV using CVV antisera and vice versa. It is, therefore better to test for each virus separately using homologous antisera.

CITRUS INFECTIOUS VARIEGATION AND CITRUS LEAF RUGOSE VIRUS DETECTION

Summary
• Graft transmission to citrus

Indicators:
CIVV -Citron, lemon (preferred), Dweet tangor, mandarin or sour orange.
CLRV - Mexican lime, Eureka lemon, Duncan grapefruit.

No. plants/test:
4 in individual containers (or planted 3 plus 1 control in each of two containers).

Inoculum:
"Buds" (buds, blind buds or chip buds).

Plant growth:
Allow full flush to develop after initial cut-back without trimming.

Temperature:
Cool: 24-27C max. day/l8-21C min. night.

First symptoms:
4 to 6 weeks.

Symptoms:
CIVV - Leaf distortion, puckered segments, epinasty and chlorotic mottle. Young leaf patterns in sweet orange, sour orange and lemon. Crinkle and puckering in mature leaves.
CLRV- Flecking in lemon, leaf puckers in Mexican lime, stunting and chlorosis in grapefruit for some strains.

• Transmission to herbaceous hosts

Indicators:
Cow pea, common bean or Crotalaria spectabilis.

No. plants/test:
8 (4 plus 1 control in each of 2 containers).

Inoculum:
Young citrus leaf tissue - tips, leaves macerated in 0.05 M phosphate buffer pH 7.0.

Inoculation:
Leaf rubbed with carborundum.

Temperature:
Cool: 20-22C.

First symptoms:
2 to 5 days.

Symptoms:
Cowpea: CIVV - Chlorotic/necrotic spots in primary leaves; systemic mottle in secondary leaves.
CLRV - Small necrotic local lesions only.
Bean (red kidney): CIVV-Brilliant vein clearing and yellow vein banding in secondary leaves. CLRV - Small necrotic local lesions only.
C. spectabilis: CIVV - Brown necrotic ringspot in primary leaves; halo, necrotic fleck and necrosis in secondary leaves.

REFERENCES

Davino, M. & Garnsey, S.M.1984. Purification, characterization and serology of a mild strain of citrus variegation virus from Florida. In Proc. 9th Conf. IOCV, p.196-203. Riverside, IOCV.

Desjardins, P.R. & Bov, J.M.1980. Infectious variegation. In Description and illustration of virus and virus-line diseases of citrus. Bov, J.M. & Vogel, R., eds. A collection of colour slides. Paris, I.R.F.A. SETCO-FRUITS.

Fawcett, H.S.1936. Citrus diseases and their control. New York, McGraw Hill. 656 pp.

Fawcett, H.S. & Klotz, L.J.1939. Infectious variegation of citrus. Phytopathol., 29: 911-912.

Garnsey, S.M.1975. Purification and properties of citrus leaf rugose virus. Phytopathol., 65: 50-57.

Garnsey, S.M. & Gonsalves, D.1976. Citrus leaf rugose virus. C.M.I./A.A.B. Descriptions of Plant Viruses, No. 164. September 1976.

Garnsey, S.M. et al.1984. A mild isolate of citrus variegation virus found in Florida citrus. In Proc. 9th Conf. IOCV, p. 188-195. Riverside, IOCV.

Grant, G.T. & Smith, P.F.1960. Infectious variegation of citrus found in Florida. Plant Dis. Rep., 44: 426429.

Majorana, G.&: Martelli, G.P.1968. Comparison of citrus infectious variegation and citrus crinkly-leaf virus isolates from Italy and California. In Proc. 4th Conf. IOCV, p. 273-279. Gainesville Univ. Fla. Press.

Majorana, G. & Scaramuzzi, G.1965. Studies on Petri's variegation of sour orange leaves. In Proc. 3rd Conf: IOCV, p. 254-259. Gainesville, Univ. Fla. Press.

Petri, L.1931. Variegatura infettiva delle foglia di Citrus vulgaris Risso. Boll. R. Sta. Patol. Veg. N.S., 11: 105-114.

Trabut, M.1913. A note. In Comptes rendus de l'Acadmie des sciences, Paris, 156: 243-244.

Uyeda, I. & Mink, G.I.1983. Relationship among some ilarviruses: proposed revision of subgroup A. Phytopathol., 73: 47-50.

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FIGURE 63 Symptoms of CIVV on leaves of navel orange from a tree in the field (Spain)

FIGURE 64 Symptoms on a citron seedling showing severe epinasty, chlorotic variegation and feat distortion induced by Inoculation with CIVV (California)

FIGURE 65 Symptoms on mature leaves of an inoculated Madame Vinous sweet orange seedling showing protuberances and bumps characteristic of infection with CIVV. These symptoms are typical and similar to those induced on mandarins or rough lemon leaves

FIGURE 66 Psorosis like symptoms In young leaves of a rough lemon seedling inoculated with CIVV

FIGURE 67 Symptoms of mild isolate CVV-2 on leaves of citron. Symptoms on lemon are quite similar (Photo: S.M. Garnsey)

FIGURE 68 Necrotic local lesions on primary leaves of cowpea infected with CIVV (Photo: S.M. Garnsey)

FIGURE 69 Chlorotic lesions on primary leaves of cowpea infected with CIVV

FIGURE 70 Symptoms on secondary growth leaves of red kidney bean infected with CIVV, showing bright vein chlorosis (Photo: S.M. Garnsey)

FIGURE 71 Symptoms on leaves of C. spectabilis infected with CIVV (Photo: S.M. Garnsey)

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