Fifteenth IOCV Conference, 2002—Short Communications

The Virus Disease Situation of in —A Brief Review

A. Kyriakou, Th. Kapari-Isaia, and N. Ioannou

ABSTRACT. Virus and virus-like diseases of citrus in Cyprus are related to the history of cit- rus on the island. , and sour were introduced to the island before the 14th century, whereas mandarin and orange were introduced in the 19th century. There are indications that all of the above citrus species came from neighboring countries of the Asian Continent. In the first half of the 20th century several citrus varieties were introduced from South Africa and Israel. In the last 50 yr, virus-free material of several varieties was introduced from California. As a result, citrus grown in Cyprus is affected by the same virus and virus-like diseases which affect citrus in neighboring countries, and by (CTV) which was most probably intro- duced with the South African material. The main virus and virus-like diseases of citrus identified so far on the island are: CTV, Citrus psorosis virus (CPsV), Citrus variegation virus (CVV), citrus impietratura, stubborn, viroid (CEVd), Citrus cachexia viroid (CCaVd) and three other citrus viroids with 284, 305 and 318 nucleotides, respectively. After CTV was detected on the island, a program was initiated for the control of the disease through systematic surveys, uproot- ing of infected trees against compensation to the growers, and production and distribution of virus-free material. Survey results from 744 groves with 406,500 trees from five showed a CTV incidence of 4.5% and a disease prevalence of 22%. Nearly all infected trees in four of the five districts were eradicated. In the fifth , however, CTV infection was high (18.3%) and eradication was not considered feasible. In addition to CTV control, the CTV Program has in gen- eral benefited citriculture in Cyprus as it paved the way for the establishment of a Citrus Certifi- cation Program.

The virus disease situation of cit- derived respectively from the San- rus in an area is often associated skrit “Nagrunga” and the Arabic with the history of citrus cultiva- “Leimun”. However, in the Greek tion. For this reason an account of Cypriot dialect, sour orange is called the history of citrus on the island “Kitromilia” (citron apple tree), indi- will be briefly outlined. All informa- cating that citron was already tion for citrus in Cyprus before the present, and lemon is “Oxinia” (from 1930s has been derived from Genna- “oxino” = acidic). Products and dius’ Botanical Dictionary (4). byproducts of these two fruits have The actual time of introduction of been traditionally used in several citrus in Cyprus is not well known, ways in cookery and confectionery. but from early accounts of the pres- Mandarins and oranges were intro- ence of citrus in the Eastern Mediter- duced much later in the second half ranean region, it is gathered that of the 19th century. The local manda- citron was present in Cyprus not rin and orange were among the later than the first century A.D, and earliest introductions. In the 1930s was probably introduced as early as citrus budwood of several varieties, the second century B.C. Sour orange, including Marsh seedless , which is the main rootstock used on Valencia and Washington Navel, the island to the present, and lemon, were introduced from South Africa. were introduced later, most probably In 1939 Valencias were introduced from , before the 14th cen- from Israel. In the 1960s nucellar tury. There is a historical account of virus-free material of several variet- the presence of a garden of sour ies was introduced from Willits & orange trees in 1468. It is worth not- Newcomb Inc., California, and in the ing that in Greece, sour orange and 1980s virus-free material of red-flesh lemon are known by the universal grapefruit and Ortanique was intro- names “Naranja” (Greek “Neranja”) duced from California and Florida. and Lemon (Greek “Lemonia”), In the last decade several varieties

427

428 Fifteenth IOCV Conference, 2002—Short Communications were introduced from California and depending on the time of year that Spain. Citrus, which originated from graft-inoculation was performed. material present on the island before Symptoms usually appeared in the 1960s, will be here designated as spring and autumn during one to ‘old-line’. two weeks and they disappeared to The first plant pathologist to work reappear on the new growth the fol- on virus diseases of citrus in Cyprus lowing spring and/or autumn. Iso- was Dr. A. Papasolomontos in the lates from the above trees reacted 1960s, who, by field symptomatology positively in ELISA tests using anti- and biological indexing, noted several sera provided by Prof. R. G. Milne, virus and virus-like diseases, includ- University of Turin, Italy (Rabbit ing psorosis, impietratura, stubborn polyclonal A322 for trapping the and tristeza. (9, 10, and pers.comm.). virus and monoclonal 13C5 for Economides (2) reported the first detection), and using the protocol for record of exocortis on old-line Marsh TAS ELISA described by Alioto et al. seedless grapefruit. (1). Positive response was obtained The present report, however, will with eight samples from the field focus on the findings of a survey for and three samples from the glass- virus and virus-like diseases which house, all with symptoms. has been conducted by the Agricul- Variegation. Variegation symp- tural Research Institute since 1986. toms have been observed on several This survey was made in the context trees of Jaffa and of a program for the production and and also on the local Lapithou distribution of clean citrus material. lemon, but repeated attempts to The main method used was the con- transmit the symptoms to indicators ventional biological indexing tech- of Citrus variegation virus (CVV), nique by the use of plant indicators including Eureka lemon and which were imported either from citron, were unsuccessful. Two iso- Willits & Newcomb or from the Uni- lates from Ortanique, however, with versity of California, Riverside. severe variegation symptoms (87- Indexing was performed in a glass- 127 and 87-128), which consisted of house with temperatures 16-33°C. tree stunting, leaf distortion and Other techniques have also been fruit reduction and malformation, used and these will be reported were transmitted by grafting and where appropriate here below. Mea- caused variegation symptoms to sures which have been taken for the Eureka lemon, Etrog citron and control of some of these diseases will Ortanique. They also caused varie- be also outlined. gation symptoms in several other The main diseases which have plant indicators, including Madam been diagnosed since 1986 are the Vinous and Pineapple sweet orange, following: Mexican , Citrus excelsa, Rough Citrus Psorosis. Classical pso- lemon and Ortanique. They were rosis symptoms of bark scaling have also transmitted mechanically to been noticed in the field on several Petunia hybrida and Eureka lemon trees of old-line Valencia and Jaffa and from this plant they were trans- orange, Marsh seedless grapefruit mitted by grafting back to Orta- and . Samples from 15 nique, thus confirming Koch’s Valencia, 11 Jaffa, four grapefruit postulates and indicating the and five Clementine trees, respec- involvement of an infectious and tively, were indexed in the glass- transmissible agent, which may be house on seedlings of Madam Vinous CVV. However, when all above and/or Pineapple sweet orange, and infected plants were tested three Dweet . Characteristic symp- times by ELISA, using antibodies toms appeared on all the indicators 6 against CVV provided by Unité de weeks to 6 mo post-inoculation, Controle des Plants, Direction de

Fifteenth IOCV Conference, 2002—Short Communications 429

Domaines Agricoles of Morocco, symptoms in the field, as over 95% of there was no reaction. It is possible citrus trees are on sour orange root- that the Cypriot isolates of CVV are stock which is tolerant to viroids. different from the Moroccan isolates. Cachexia. This disease has been Impietratura. This disease was observed mainly on old-line Clemen- first reported by Papasolomontos tines, but also on some local manda- (12) and affects some old-line grape- rins and on Ortanique. Trees of fruit plantings, but was also found these varieties with the characteris- in some Jaffa, Valencia and acidless tic field symptoms have indexed oranges. Usual symptoms consist of positive for cachexia on Parson’s gumming of the fruit albedo, hard- special mandarin budded on Rough ening of the fruit and reduction of lemon or . In 1994 fruit size. It has been observed that cachexia viroid was also identified symptoms of impietratura are more by electrophoresis and was found serious on young trees, and that present in the local lemon and in symptoms on trees over 12-yr old Valencia and Jaffa oranges. are less conspicuous and inconsis- Stubborn. The disease has been tent from year to year. However, two noted in Cyprus since 1956 by isolates of the disease from Valencia Economides (pers. comm.), but was caused consistently severe fruit known to growers even earlier with symptoms and intense psorosis-like the local name “psintrophyllia” (=lit- symptoms on leaves of sweet orange tle leaf). The causal agent of stub- in the field and the glasshouse. The born, Spiroplasma citri, was problem needs further investigation. isolated in culture from Valencia Citrus Exocortis and Related and Washington navel orange trees Viroids. Exocortis disease was first with typical disease symptoms in reported in Cyprus by Economides 1987 (Bové, Saillard, Foss and Kyri- (2) during a scion-rootstock trial, in akou, unpublished data). which old-line Marsh seedless grape- A study conducted at the experi- fruit was budded on Morton and mental station of the Agricultural Troyer . During the virus Research Institute in survey which was conducted later (7), showed that stubborn had a serious it was found that of 573 trees of 25 adverse effect on fruit yield, size and citrus species and varieties sampled, quality of 12-yr-old Washington 506 were found to be infected when Navel and Frost Valencia oranges. indexed by grafting to Etrog citron Fruit yield was reduced by 19-34% indicators. Over 90% of the samples and fruit weight and diameter by which indexed positive for viroids 29% and 12%, respectively, when produced severe symptoms on citron, compared with fruit from healthy indicating the wide distribution of trees. Total acidity was higher and Citrus exocortis viroid (CEVd). In the ratio of total soluble solids to 1994 electrophoresis on citron sam- total acids was lower in infected ples with mild symptoms, which had fruits of both varieties (10). It was been obtained by biological indexing later observed however, that when of micrografted samples of the local trees grew older, the effect of stub- “Lapithou” lemon (5), revealed the born decreased and symptoms presence of three other viroids: the became less conspicuous, but the citron chimera viroid with 284 nucle- systematic study was not repeated. otides, a viroid with 305 nucleotides Tristeza. Citrus tristeza virus and Citrus bent leaf viroid with 318 (CTV) was first detected in Cyprus nucleotides (Hoffman, Kyriakou, by Papasolomontos and Econo- Hadjinicolis and Bar-Joseph, unpub- mides (13) when 27 trees of five cit- lished data). Citrus exocortis and rus species were found infected and related viroids, although widely dis- destroyed. During the latest virus tributed, do not produce conspicuous survey which has been conducted

430 Fifteenth IOCV Conference, 2002—Short Communications since 1986, CTV was initially practice of removal of infected trees detected in four out of 156 groves was applied, however, in the other surveyed by the use of Mexican lime areas, with compensation to the indicators (8, 9) In view of the dan- growers. Approximately 4,000 trees ger of an epidemic spread of CTV have been uprooted to the present, and the prevalent use of the CTV- including six entire groves. sensitive sour orange rootstock, a Field symptoms of CTV-infected project for the control of this disease trees varied from inconspicuous to was initiated in 1992 (6, 11) The severe. The most intense symptoms basic objectives of the project are; a) were noted on Marsh seedless and the mapping of CTV infection Star Ruby grapefruit, and included through a systematic survey of cit- stunting, chlorosis, fragility and die- rus, b) the removal of infected trees back of twigs, pitting of branches or groves where this is feasible and general decline. Tristeza caused against compensation to the grow- severe decline and death of 40-50 yr- ers, and c) the establishment of a old grapefruit and Valencia orange viable citrus certification program. trees in certain groves in the dis- The survey was conducted by tricts of and Lemessos, indexing, using ELISA, 10-20% of respectively. With regard to the the trees in each grove in the five greenhouse indexing tests, usually citrus-producing districts of Cyprus. the intensity of field symptoms Several CTV isolates were grafted related well to the severity of CTV on Mexican lime, sweet orange, sour symptoms on Mexican lime in the orange and grapefruit in a green- greenhouse. No seedling yellows house. Results of the survey showed symptoms on sour orange or grape- an average CTV incidence of 4.46% fruit were observed. (Table 1). From 58,127 trees indexed There are strong indications that and obtained from 744 groves with CTV in Cyprus has been spread 406,500 trees, 2,592 trees were mainly through infected budwood found to be CTV-infected. Of the 744 and that the major source of CTV groves surveyed, 164 had CTV- was infected material introduced infected trees. Disease incidence from South Africa in the 1930s and and prevalence ranged in the differ- established in a citrus nursery in ent districts from 2.7% to 18.3% and the where the from 17.9% to 72.4%, respectively. highest disease incidence was noted The highest proportion of infected (6). The proportion of infection in trees and groves was noted in the the other areas shows that control of district of Famagusta (Table 1), CTV is still possible by removal of where it was decided that eradica- infected trees and use of virus-free tion was no longer feasible. The planting material for the establish-

TABLE 1 SURVEY FOR CITRUS TRISTEZA VIRUS IN FIVE DISTRICTS OF CYPRUS, 1992-2001

Districtz

Description Famagusta Paphos Total

No. groves indexed 392 29 180 49 94 744 No. groves infected 70 21 39 17 17 164 % groves infected 17.86% 72.41% 21.67% 34.69% 18.09% 22.04% No. trees indexed 26,060 1,514 20,519 3,696 6,338 58,127 No. trees infected 1,016 277 552 272 475 2592 % trees infected 3.90% 18.30% 2.69% 7.36% 7.49% 4.46% No. trees in groves indexed 96,855 3,846 205,087 32,184 68,528 406,500

ZIndexing was by enzyme-linked immunosorbent assay (ELISA).

Fifteenth IOCV Conference, 2002—Short Communications 431 ment of new groves through a man- groves established before the 1960s, datory certification program (3). as certified citrus material, which Efforts are made to clear the infected was disseminated in the island later, area of Famagusta from trees with was free of them. The viroids, which severe CTV isolates. This will dimin- are transmitted both by grafting and ish the danger of dissemination of mechanically, are widespread, but do severe CTV isolates in case Toxop- not always cause obvious symptoms tera citricida invades the Mediterra- because of the tolerance to viroids of nean basin. sour orange, the main citrus root- stock. The most serious virus disease CONCLUSIONS faced by citriculture in Cyprus is tristeza, for the control of which a The citrus virus and virus-like special program has been imple- disease problems of Cyprus are simi- mented since 1992. The low CTV lar to those of neighboring Mediter- incidence in most areas of Cyprus ranean countries and include allows control of the disease by psorosis, impietratura, variegation, removal of infected trees and estab- stubborn, exocortis, cachexia and lishment of new groves with virus- other viroids. The first three dis- tested material through the applica- eases affect almost exclusively citrus tion of a certification program.

LITERATURE CITED

1. Alioto, D., M. Gangemi, S. Deaglio, P. Sposato, E. Noris, E. Luisoni, and R. G. Milne 1999. Improved detection of citrus psorosis virus using polyclonal and monoclonal anti- bodies. Plant Pathol. 48: 735-741. 2. Economides, C. V. 1976. Exocortis in Marsh seedless grapefruit in Cyprus. Plant Dis. Reptr. 60: 532-534. 3. Gavriel, I. 2002. The citrus certification program of Cyprus. In: Proc. 15th Conf. IOCV, 420-222. IOCV, Riverside, CA. 4. Gennadius, P. G. 1959. Botanical Dictionary, Moskhou Giourdas, Athens, Greece, 295-354, 1041 pp. (in Greek). 5. Ioannou, M., A. Kyriakou, and N. Ioannou 1991. Production of healthy by shoot-tip grafting in vitro. Tech. Bull. No. 139, Agric. Res. Inst., Min. Agric. & Nat. Res., Nicosia, Cyprus, 7 pp. 6. Kapari, Th., A. Kyriakou, N. Ioannou, J. Gavriel, M. Bar-Joseph, G. Savva, D. Polycarpou, N. Loizias, E. Iosephidou, A. Hadjinicolis, and Chr. Papayiannis 2000. A six-year fight against citrus tristeza virus in Cyprus. In: Proc. 14th Conf. IOCV, 335-337. IOCV, Riverside, CA. 7. Kyriakou, A. P. 1992. Incidence in Cyprus of citrus exocortis viroid and its mechanical transmission. Plant Pathol. 41: 20-24. 8. Kyriakou, A. and D. Polycarpou 1989. Detection of citrus tristeza virus by indexing. FAO Plant Prot. Bull. 37: 131-132. 9. Kyriakou, A., D. Polycarpou, A. Efstathiou, and A. Hadjinicoli 1992. Citrus tristeza virus in Cyprus. In: Proc. 12th Conf. IOCV, 69-72. IOCV, Riverside, CA. 10. Kyriakou, A., G. Eliades, N. Ioannou, and Th. Kapari-Isaia 1996. Effect of stubborn disease on growth, yield and fruit quality of Frost Washington Navel and Frost Valencia oranges in Cyprus. J. Hort. Sci. 71: 461-467. 11. Kyriakou, A., N. Ioannou, J. Gavriel, M. Bar-Joseph, Chr. Papayiannis, Th. Kapari-Isaia, and G. Savva 1996. Management of citrus tristeza virus in Cyprus. In: Proc. 13th Conf. IOCV, 172- 178. IOCV, Riverside, CA. 12. Papasolomontos, A. 1961. Preliminary report on a new disorder on grapefruit. Agrotis, Nicosia, Cyprus. 13. Papasolomontos, A. and C. V. Economides 1968. The presence of tristeza virus in certain species of citrus in Cyprus. FAO Plant Prot. Bull. 16: 8-9.