Management of Citrus Tristeza Virus in A. Kyriakou, N. Ioannou, J. Gavriel, M. Bar-Joseph, Chr. Papayiannis, Th. Kapari-Isaia, and G. Savva

ABSTRACT. Following the detection of citrus tristeza virus (CTV) in Cyprus, a 5-yr program was initiated in 1992 to control the disease. The basic objectives of this program are (i) the survey of all citrus to assess the actual CTV incidence and spread, (ii) the removal of infected trees wher- ever it is feasible, and (iii) the production and distribution of healthy propagating material of cit- rus through the development of a Budwood-Certification Program. The results to date from five citrus-producing showed an average disease incidence of 5.8%. Thus, of the 26,678 trees indexed by ELISA and representing 335 groves with a total of 126,519 trees, 1,557 trees of at least 12 species and/or varieties were found to be CTV-infected. Prevalence of CTV among groves was 23% (791335). In four of the five districts surveyed, all of the CTV-infected trees have been removed or are in the process of removal and growers will be compensated. However, in the fifth district where infection was high (18.3%), it was decided that eradication of the disease was no longer feasible. In order to minimize the danger of transmission of CTV from this area to other areas, the transfer of citrus planting material to other parts of the island was prohibited by quarantine rules. For the production and distribution of CTV-free budwood in Cyprus, sanitation measures were enforced in all private nurseries. Concurrently, efforts are being made through appropriate legislation for the establishment of a Citrus Certification Program.

Citrus is a major crop in Cyprus will prevent the introduction of CTV and occupies 7,500 ha mainly in the to areas where eradication has taken coastal plains and in one area of the place. It will also minimize the dan- central plain of . A prelimi- ger from the introduction and spread nary survey in 1987 revealed the of new virulent strains of CTV. presence of citrus tristeza virus Current results of the project for (CTV) in several citrus trees grown the control of CTV which have been in four of 156 groves sampled (3). In obtained to the present time and the view of the widespread use of the Citrus Certification Program are CTV-sensitive sour orange rootstock described. in the island and the destructive spread of CTV in other Mediterra- MATERIALS AND METHODS nean countries (4), a 5-371- national project was undertaken in 1992 for Survey and indexing for CTV. the early control of CTV in an effort Samples were taken from 10-20% of to avoid the possible losses to the cit- the trees within each grove, each rus industry due to epidemic spread sample consisted of four 10 cm long of severe isolates of the virus. This twigs obtained from the four sides of Ministry of Agriculture Project each tree. They were placed individ- involves a cooperative effort between ually in plastic bags and processed two of its departments, the Agricul- the following day. For the first two tural Research Institute and the years, the samples were ground in Department of Agriculture. The pro- buffer using a Kleco tissumizer and gram has two main objectives: (i) then tested by DAS ELISA in Nunc systematic survey of all citrus to microplates against monoclonal assess CTV incidence and spread, antibodies purchased from Immuno- and (ii) removal of infected trees logia y Genetica Applicada, S.A., andlor groves where this is feasible. Spain. However, in the last year the In conjunction with this project, samples were cut directly into 2-3 efforts are also being made to estab- mm pieces in buffer-containing wells lish a Certification Program which of precoated microplates and tested Thirteenth ZOCV Conference, 1996- Citrus Tristeza Virus 173 by indirect ELISA against polyclonal ble for a citrus foundation block antisera obtained from the which will be established in an Tolkowsky laboratory, Bet Dagan, insect-proof screenhouse of about Israel (1). One well was used per 350 m2at the experimental station of sample and each ELISA plate con- the Institute at Akhelia, on the west tained one negative and two positive coast of the island (Fig. 1).The foun- controls, one from the greenhouse dation block will house all citrus spe- and one from the field. If CTV was cies andlor varieties presently detected in any sample from a grove, available in Cyprus as virus-free then all the trees in that particular material. Each accession will be grove were tested in order to identify maintained in two plants propa- all infected trees. gated on sour orange and Volkameri- Several CTV isolates from the dif- ana rootstocks. ferent varieties and various loca- The Department of Agriculture tions were grafted to Mexican lime will be responsible for the mother seedlings in a controlled-tempera- and budwood increase blocks. An ture glasshouse (15-32'0, for the isolated open-field mother block will establishment of a CTV isolates col- be established at , a few kilo- lection. Biological characterization meters away from commercial cit- of these isolates was done in the fol- rus groves and will include 4-5 trees lowing hosts: sour orange, Washing- per accession propagated on sour ton Navel orange, Star Ruby orange and Volkameriana root- grapefruit and Eureka lemon. stocks. One budwood increase block Foundation block and bud- under insect-proof screen of about wood increase blocks for Certifi- 300 m2 has already been established cation Program. The Agricultural and another similar one will soon be Research Institute will be responsi- constructed. This will be used to

Fig. 1. Survey of citrus in five districts of Cyprus for citrus tristeza virus. Sites (com- munities) covered by the survey indicated by black dots. : , , Polis, Prodromi, , Paphos, Koloni, Akhelia, Agia Varvara, Timi, Mandria, Kouklia. Nicosia: Pakhyammos, , , Eurykhou, Koutraphas, , , , Avlona, Akaki, Potamia. Limassol: Avdimou, Episkopi, Erimi, Kolossi, Ypsonas, Trakhoni, Akrotiri, Polemidia. : Agios Theodoros, Mosphiloti, , Xylophagou. Famagusta: , Vrysoules, . 174 Thirteenth IOCV Conference, 1996-Citrus Tristeza Virus increase the number of buds avail- dieback and general tree decline. able for propagation of certified Some old Valencia trees appeared plants of certain popular varieties. stunted and produced small fruit. In Buds will be collected from these the glasshouse the degree of symp- blocks for a maximum period of 5 tom severity on Mexican lime related well to the intensity of field symptoms. Isolates from mildly RESULTS affected trees produced mild vein clearing and light stem pitting on Survey. Results obtained from Mexican lime indicators, whereas the five main citrus-producing dis- samples from seriously affected tricts of Cyprus (Fig. 1)are shown on trees produced severe vein clearing, Table 1. From a total of 335 groves stunting, leaf cupping and stem pit- which included approximately ting. A preliminary biological char- 127,000 trees all on sour orange acterization of several CTV isolates rootstock, infection was found in 79 did not reveal any seedling yellows groves (disease prevalence = 23%) isolates, but a few isolates caused and average disease incidence was stem pitting on Star Ruby grape- 5.8%. Infection with CTV was found fruit, while a single isolate (89-197) in nearly all citrus species or variet- caused pitting on sour orange and ies indexed, with the highest inci- Washington Navel sweet orange. dence being noted on clementine, Certification. Presently the grapefruit (particularly Star Ruby) foundation block includes the follow- and Ortanique (Table 2). ing citrus accessions: (a) 11 varieties Field and glasshouse symp- which were imported as nucellar or toms. Trees infected with CTV as shoot-tip grafted material from exhibited various field symptoms. the University of California, River- Many infected trees were symptom- side, during different periods since less, but several showed mild to 1979; (b) four varieties which were severe decline. Some infected clem- imported from MA, Valencia in entine and Ortanique trees exhib- 1993; and (c) six clones of the local ited mild chlorosis, stunting and lemon variety "Lapithos" which were occasionally twig dieback. Young produced locally by shoot-tip graft- Star Ruby grapefruit showed ing in vitro (2). All this material was stunted growth and mild stem pit- found free of known virus and virus- ting, whereas 30-year old Marsh like diseases after being biologically Seedless grapefruit showed pitting indexed following the techniques of branches and trunk, twig fragility, described by Roistacher (7). In addi-

TABLE 1 SURVEY FOR CITRUS TRISTEZAVIRUS IN FIVE DISTRICTS OF CYPRUS, 1992-1995'

District

Description Nicosia Famagusta Limassol Larnaca Paphos Total

No. of groves indexed 163 29 70 36 37 335 No.of groves infected 33 21 9 13 3 79 % of groves infected 19.0 72.4 12.9 36.1 8.1 23.0 No. of trees indexed 12,502 1,514 7,052 2,683 2,927 26,678 No. of trees infected 588 277 54 254 384 1,557 % of trees infected 4.6 18.3 0.8 9.5 13.1 5.8 Total no. of trees included 59,180 6,374 38,035 8,983 13,947 126,519 in the groves indexed

zIndexingwas by enzyme-linked immunosorbent assay (ELISA) Y 5 P $ 8 TABLE 2 2 INCIDENCE OF CITRUS TRISTEZA VIRUS IN DIFFERENT CITRUS SPECIES AND/OR VARIETIES IN FIVE DISTRICTS OF CYPRUS, 1992-1995' 9 District 32 3 Citrus species or variety Nicosia Famagusta Limassol Larnaca Paphos Total hr Lemon 2311406~ 24/78 61451 33198 41113 9012146 $o, Valencia orange 3413558 61127 01298 11403 01127 4114513 I Jaffa orange 121303 231338 41168 11540 01106 4011455 F9 Shekeriko orange 0183 0175 011 1 0138 01207 E Navel orange 01404 41423 4180 1/58 91965 ? Marsh Seedless grapefruit 2911757 1911816 011044 31278 016 22313901 Z' Star Ruby grapefruit 2801689 411297 01154 01263 28412403 f Mandarin 31283 4/45 0150 0126 0139 71443 Clementine 191459 013 1 141493 2081495 01144 24111658 S Ortanique 17312627 1712436 21437 37711969 56917469 E Sour orange 4140 012 213 3 011 6/76 Bergamont 212 7 111 0146 0117 0115 31106 Miscellaneous 91830 28/76 31238 21144 2/48 4411336 Total 572112502 27711514 5417052 25412683 38412927 1557126678 Percentage % 4.6 18.3 0.8 9.5 13.1 5.8

%Indexingwas by enzyme-linked immunosorbent assay (ELISA) ?Values indicate the number of CTV-positive treesltotal number of trees tested 176 Thirteenth IOCV Conference, 1996-Citrus Tristeza Virus tion to the above material. the foun- Eradication is mandatory (legal reg- dation block may include 16 citrus ulation 246/90), and compensation accessions which were introduced per tree was provided according to a recently from USDA, Riverside, and prescribed formula which is based are presently maintained in a quar- on the variety, the age and the pro- antine greenhouse of the Depart- ductivity of the tree with an average ment of Agriculture where indexing value of US $24 per tree. for known citrus viruses is ongoing. The district of Famagusta (Fig. With regard to commercial nurs- I), which had the highest disease eries, during the survey for CTV, incidence, is the oldest citrus grow- infection was noted in mother trees ing area of the island. It is probable and young grafted trees in two out of that tristeza was disseminated in eight private nurseries tested. All this area with infected material infected material was destroyed and which was introduced from South nursery men were requested to Africa in 1929 and established in an obtain in the future their propagat- experimental grove located in this ing material from the Department of district (6). Some of this infected Agriculture till regulations for nurs- material was probably carried to the ery operation are fully enforced. other citrus growing areas. However, the majority of plant material used DISCUSSION to establish citrus in these areas came from available propagating Plant pathologists have suc- budwood, or were introduced period- ceeded in convincing the authorities ically from California since the in Cyprus that action must be taken 1960's. to control CTV. As a result, funds To minimize the danger for trans- were secured in 1992 for a special 5- mission of CTV from Famagusta to year project aiming at a systematic the other districts, a legal regulation survey of citrus for CTV and the was issued (131193) which forbids eradication of infection foci. the transport of citrus planting Although results obtained so far material from this district to other show that tristeza is more wide- areas. However, efforts are also spread than initially thought, it being made to apply eradication seems that eradication is still feasi- measures to groves or individual ble in four of the five districts sur- trees which are infected with severe veyed. tristeza isolates. For example one The role of aphid vectors in the grove of about 500 grapefruit trees dissemination of CTV inside Cyprus infected with stem pitting isolates is not known yet, although Aphis has already been removed. In all gossypii was shown experimentally other districts all known individual to be an efficient vector of the virus infected trees, as well as three (3). There are some indications that groves were removed by June 1996 presently CTV is not efficiently and growers were compensated. transmitted in field plantings. How- The higher incidence of infection ever, a systematic epidemiological found in some districts in Ortanique study needs to be conducted to deter- and Star Ruby grapefruit (Table 2), mine the extent of natural spread of is notable. These two varieties were CTV. nucellar introductions from the In those cases where percentage 1970's from the United States. It of infected trees was 15% or higher, now appears that some of these two it was recommended that the whole varieties were either (i) introduced grove be destroyed. When CTV inci- from other foreign-infected sources dence was lower it was suggested or (ii) top-grafted on older CTV- that only infected trees be removed. infected trees by growers and/or Thirteenth IOCV Conference, 1996- Citrus Tristeza Virus 177 nurseries and were disseminated tion block and field mother blocks from there. will be indexed yearly for CTV by These incidents are compelling ELISA, every three years for viroids, and justify the development of a and every six to ten years for other sound Certification Program. This diseases including psorosis, infec- necessity has been officially recog- tious variegation, concave gum, nized with the passing of relevant impietratura, cristacortis, tatter- legislation and the approval of funds leaf and woody gall by biological for the initiation of a Certification indexing following the techniques Program similar to that operating in described by Roistacher (7). They Spain (5). will also be inspected visually once a Distribution of citrus mate- year for identification of trees with rial by the state. The Department symptoms of stubborn or with other of Agriculture, which covers about horticultural abnormalities. Precau- 50% of the need of the growers' mar- tion will be taken to control pests ket for citrus seedlings and young and fungal diseases. Citrus material trees, will establish the production of in budwood increase blocks and com- all citrus planting material under mercial nurseries will be inspected screen to minimize the risk of infec- annually for fungal and physiologi- tion by CTV and other vector-trans- cal problems and will be sampled mitted diseases, such as stubborn. and tested for CTV, as well as other Commercial nurseries. Legal virus and virus-like pathogens. regulations which were issued in Due to the small size of the 1994 (50194), on the basis of a law island, and the limited number of (60191) for the production and distri- private citrus nurseries, the produc- bution of healthy planting material, tion and distribution of healthy cit- provide details of the prerequisites rus material throughout Cyprus is for registration of the nurseries and expected to become a reality within a the production and release of few years, as the research and exten- healthy citrus material. The com- sion services continue their work mercial nurseries have to obtain and secure the cooperation of the clean material either as budwood or private nurseries. as young budded trees from the ~epartmentof Agriculture and they may establish their own multiplica- ACKNOWLEDGMENTS tion block which can be kept for no longer than five years. In addition, We thank Christina Triantaphyl- all citrus material, seedlings, young lidou, Georgios Psiloinis, Demetris grafted trees and multiplication Polycarpou, and Nicos Loizias for blocks will have to be kept under their valuable assistance in mapping insect-proof screen. The government groves and collecting and processing will assist those nursery-men who samples. The assistance of Artemis are interested in registering their Hadjinicoli and Andreas Hadjinicolis nurseries with low-interest loans for is gratefully acknowledged. We are construction of suitable screen- finally indebted to Dr. Andreas houses. Papasolomontos and Dr. Panayiotis Indexing and visual inspec- Orphanos for critically reviewing the tions. All plants in the basic founda- manuscript.

LITERATURE CITED

1. Bar-Joseph, M. and A. Hadjinicolis 1994. Sensitive detection of citrus tristeza virus by dipping and incubation of stem seg- ments in ELISA wells. Hassadeh 74: 76 (Hebrew with English abstract). 178 Thirteenth IOCV Conference, 1996- Citrus Tristeza Virus

2. Ioannou, M., A. Kyriakou, and N. Ioannou 1991. Production of healthy Lapithos lemon plants by shoot-tip grafting in vitro. Techni- cal Bulletin 139, Agricultural Research Institute, Nicosia, 7 pp. 3. Kyriakou, A., D. Polycarpou, A. Efstathiou, and A. Hadjinicoli 1993. Citrus tristeza virus in Cyprus, p. 69-72, In: Proc. 12th Conf. IOCV., IOCV, River- side. 4. Moreno, P., J. Piquer, J. A. Pina, J. Juarez, and M. Cambra 1988. Spread of citrus tristeza virus in a heavily infested citrus area in Spain, p. 71-76, In: Proc. 10th Conf. IOCV., IOCV, Riverside. 5. Navarro, L. 1993. Citrus sanitation, quarantine and certification programs, p. 383-391, In: Proc. 12th Conf. IOCV., IOCV, Riverside. 6. Papasolomontos, A. and C. V. Economides 1968. The presence of tristeza virus in certain species of citrus in Cyprus. FA0 Plant Protection Bull. 16 (1): 8-9. 7. Roistacher, C. N. 1991. Graft transmissible diseases of citrus. Handbook for detection and diagnosis. FAO, Rome, 286 pp.