Epidemiology of in the Old World

J. M. Bove, A. Fos, J. Lallemand, A. Raie, Y. Ali, N. Ahmed, C. Saillard and J. C. Vignault

ABSTRACT. Natural transmission of Spiroplasma citri, the causal agent of citrus stubborn disease, to periwinkle plants has been experimentally documented in Morocco and Syria. have been collected in Morocco, Turkey, Syria and France (Corsica) and analyzed for infection with S. citri. Neoaliturus (Circulifer) haematoceps was the only species found to be naturally infected with S. citri in Morocco, Syria, France (Corsica) and Iraq. N. haematoceps was reared in Bordeaux and shown to be able to acquire S. citri by feeding on S. citri-infected plants, to support multplication of the spiroplasma in its body and to transmit the stubborn agent to healthy periwinkles. Leafhoppers injected with a pure culture of S. citri were also able to infect healthy periwinkles. These experiments show that N. huematoceps is a vector of S. citri. In view of the paucity of N. tenellus in the Mediterranean area, N. haematoceps seems to be the major vector of S. citri. In Iran, N. haematoceps is also the major species, but N. tenellus seems to be more prevalent than in the Mediterranean area. Hence, in the Middle East, both N. haematoceps and N. tenellus can be vectors. Host plants of N. haernatoceps have been identified in Syria and France (Corsica). Their occurrence along the Mediterranean coast explains recent epidemics in Syria. Their very wide distribution throughout Iran, explains the presence of N. huematoceps in or close to all major citrus growing areas. Natural transmission of S. citri in Iran has been documented and explains the presence of stubborn disease on local sweet orange trees as well as on Iranian nucellar trees. However, importa- tion of S. citri-infected budwood from California or Mediterranean countries to Iran cannot be ruled out. Index words. Stubborn, Spiroplasma citri, Neoaliturus haematoceps, Leafhopper-vector, Leafhop- per host plants.

INTRODUCTION: DISTRIBUTION AND found to be transmitted vertically NATURAL SPREAD OF through seeds, even though the seeds CITRUS STUBBORN DISEASE coats from infected citrus trees can carry S. citri. Spiroplasma citri is the causal Citrus is not the only host plant of agent of citrus stubborn disease S. citri. Many non-rutaceous hosts (CSD). On the basis of symptomatol- can become infected experimentally ogy, CSD extends throughout the Old or naturally (9). Ornamental World from Morocco on the Atlantic periwinkle seedlings were the first to Iran beyond the Caspian sea and such hosts to be found naturally in- the Gulf of Oman. Our group has cul- fected with S. citri, first in Arizona tured S. citri from citrus trees in the and California (1, 12), later in following countries during various Morocco (4). More recently we have surveys: Morocco, Algeria, France cultured S. citri from ornamental (Corsica), Turkey, Syria, Israel, Jor- periwinkle seedlings collected in dan, Saudi Arabia and Iran. The wide Dubai' (United Arab Emirates) and distribution of CSD in the Old World Nizwa (Sultanate of Oman). is not only due to the use of infected Periwinkle seedlings are good indi- budwood; natural transmission has cator plants for S. citri infection, and been suspected long ago from the fact they have been used experimentally that citrus seedling trees sometimes to gain information on natural trans- show symptoms of CSD. The occur- mission of the spiroplasma. By setting rence of stubborn symptoms on seed- up periwinkle beds in various loca- ling trees or plants is taken as evi- tions, we have shown that S. citri is dence for natural spread of the dis- naturally transmitted in Morocco and ease because S. citri has never been Syria (5, 6, 7). Tenth ZOCV Conference

THE LEAFHOPPER leafhoppers collected in Palmyra NEOALITURUS HAEMATOCEPS (Syria), was used in the transmission IS NATURALLY INFECTED studies. Leafhoppers injected with WITH S. CITRI the Palmyra isolate were able to mul- tiply the spiroplasma to titers of lO5- In 1976, the leafhopper lo6 CFU per , and to transmit Neoaliturus (Circulifer) tenellus it to healthy periwinkle indicator (Baker) was identified as a major vec- plants, which began to show tor of S. citri in California (13). symptoms 6 weeks after infection. N. This discovery led us to search for haematoceps leafhoppers were also N. tenellus in Morocco where its pres- able to acquire S. citri by acquisition ence had been reported previously feeding on periwinkle plants previ- (11). In spite of many attempts, not ously infected with the Palmyra iso- only in Morocco but also in Turkey late, and to transmit it to periwinkle and Syria, N. tenellus was found in plants. The efficiency of transmission such low numbers that it did not ap- seems high since 60% of individual pear as a major vector of S. citri in leafhoppers were able to transmit the the Mediterranean area. We thus spiroplasma to periwinkles after a la- looked for alternative leafhopper vec- tent period of 20 days on stock (10). tors. Leafhoppers were collected with Both male and female individuals a D-vac aspirator, pooled according to transmit S. citri. No evidence for ver- species and analyzed for the presence tical transmission has been obtained. of S. citri by ELISA (16) and the cul- In previous experiments, we ture assay (3). Over 50 leafhopper failed to get transmission with the species have been analyzed in this leafhopper Euscelidius variegatus way on several surveys in Morocco, (Kirschbaum), the S. citri isolate used Syria, Turkey, France (Corsica) and being a strain (Israel) that had been Iraq between 1978 and 1986. Only one maintained by graft inoculations in leafhopper species was found in all periwinkles for over 10 yr. We have five countries to be naturally infected recently examined the ability of N. with S. citri, namely Neoaliturus haemematoceps to transmit the Israel haematoceps (Mulsant & Rey). In- and the Palmyra isolates of S. citri. terestingly, this species is absent The spiroplasmas were used in paral- from the new world, but is very simi- lel experiments. The spiroplasma cul- lar to the North American vector, N. tures had the same low passage num- tenellus; the two species can only be bers. No transmissions were obtained distinguished on the basis of their after acquisition by feeding or injec- male genitalia. Of all Neoaliturus tion with the Israel isolate, whereas species encountered in the Mediterra- high transmission rates were ob- nean area, N. huematoceps was the served with the Palmyra isolate. most abundant, as already reported These experiments seem to indicate earlier (11). that S. citri loses the ability of being N. HAEMATOCEPS IS A transmitted by leafhoppers when it is VECTOR OF S. CITRI kept too long in plants without under- going a cycle through the insect. The Natural infection of N. loss of transmissibility is independant haematoceps with S. citri prompted of pathogenicity to plants, since the us to investigate the potential of this Israel isolate continues to induce se- species to act as a vector. S. citri- vere symptoms in plants. These ob- free leafhoppers were collected in servations might indicate that old, Corsica and raised in Bordeaux on or- symptomatic "stubborn" trees do not namental stock (Matthiola incana function as sources of S. citri in- L.). An isolate of S. citri, cultured oculum for transmission by N. from a pool of N. haematoceps haematoceps. Diseases Induced by Procaryotic Pathogens

S. KALI, A MAJOR them infected with S. citri, were col- HOST PLANT OF lected on a few ornamental stock N. HAEMATOCEPS plants in the Orontes valley, a sugar beet, but not a citrus, region. In Cor- One of the major results of the sica, the leafhopper overwinters on work carried out in Syria was the dis- wild stock. covery of the Chenopodiaceae, Sal- N. haematoceps was also collected sola kali L., as a major host plant of on Alhagi camelorum along the Sy- N. haematoceps. Interestingly, this rian coast where this legume often oc- plant, also named S. pestijier (Russian curs in association with S. kali. In thistle, tumbleweed), is a major host southeast Iran, A. camelmm and A. of N. tenellus, the principal vector of persarum can be seen within citrus S. citri in California and Arizona. In orchards. the Old World the natural distribution Finally, plants of the typical of S. kali covers the Mediterranean Mediterranean vegetation "maquis" zone and most of the Irano-Turanian harbor N. haematoceps in Corsica (8). regions. It has spread over all arid and semi-arid areas in the warm tem- perature zones. The species is ex- EPIDEMICS OF STUBBORN tremely common in Morocco, Syria, DISEASE IN SYRIA Iraq and Iran, particularly on dis- In the Mediterranean area, stub- turbed habitats (ruderal places in born disease was first reported in towns, villages, road sides), on irri- Palestine in 1931 by Reichert and gated and non-irrigated arable land, Perlberger (15), who indicated that and on seashores; it extends from the the disease was in an epidemic form hot lowlands up to more than 2,000 and causing serious damage to young m, with highest densities in the hot citrus orchards. More recently, in the and arid to semi-arid lowlands (Prof. Ghor (Jordan River valley), young cit- Helmut Freitag, personal communi- rus plantings, established in a noncit- cation). In Syria, S. kali is very com- rus growing area, showed a high inci- mon along the seashores and the coas- dence of stubborn infection (M. Bar- tal citrus areas. Both S. citri infected Joseph, personal communication). and uninfected N. haematoceps Epidemics of stubborn are also known leafhoppers are frequently collected in California where they occur in cer- on S. kali plants. In Iran, not only N. tain years, 1973 for instance. A stub- haematoceps but also N. tenellus born epidemic has occurred in Syria occur, even though the latter is much at El Annadeh, south of Lattaquia less widespread than the former. The and at Tartous in the late 1970's, and distribution of these leafhoppers was responsible for infection with S. throughout Iran correlates well with citri of many young citrus trees from the presence of S. kali. In Iraq, S. two important nursery projects. The kali was the only plant on which N. Syrian epidemic has been the incen- haematoceps, with and without S. tive to a FA0 project to study natural citri infection, could be collected. In transmission of S. citri in Syria. Work Cyprus, S. kali is very frequent along carried out from 1982 to 1985 has the southern shores and a search for shown that the nurseries were estab- Neoaliturus sp. is underway. The lished in areas along the coast where situation in Corsica, where S. kali two major host plants of N. also occurs on the coast, is described haematoceps occur: Salsola kali and elsewhere (8). Alhagi camelorum. S. citri-infected Besides S. kali, ornamental stock N. haematoceps leafhoppers are fre- and wild stock (M. sinuata L.) are quently collected on these hosts. The good cruciferous hosts of N. land for the nurseries was cleared in haematoceps. In Syria, hundreds of 1978 and the natural vegetation in- N. haematoceps leafhoppers, most of cluding S. kali and A. camelorum, 298 Tenth ZOCV Conference was removed. This left barren, sandy haematoceps-N. tenellus, the pres- land with no windbreaks around the ence of S. kali and other major host plots to be planted with citrus. The plants of the leafhoppers and the dis- young trees that were planted in this tribution of CSD in the major citrus open land probably served as alter- growing areas of Iran. nate host plants on which leafhoppers were practically forced to feed. In CONCLUSION short, it is believed that land clearing operations under semi-desert condi- Natural spread of CSD has been tions favored leafhopper migration demonstrated in several countries of and feeding on young trees resulting the Old World. Neoaliturus in local epidemics. At the El Annadeh haematoceps has been identified as a project, new land was prepared in major leafhopper vector of S. citri. 1985 to extend the nursery. In June This species is related to N. tenellus, 1986, Salsola kali was growing again a major vector in the southwestern amid the young nursery trees and will USA. The biology of the two leafhop- probably help to attract N. pers is similar. While N. haematoceps haematoceps for a new epidemic. does not occur in the New World, N. tenellus is present in the Old World. STUBBORN IN IRAN In the Mediterranean area, N. tenel- lus does not seem to be a major vector In Iran, CSD is widespread in view of its scarcity. In Iran, it throughout the citrus growing areas. might play a larger role. Major host The first S. citri isolate from an Ira- plants of N. haematoceps have been nian citrus sample was cultured in identified. Some of these, such as Sal- Bordeaux as early as 1974. Since sola kali, are also major hosts of N. then, S. citri was isolated from citrus tenellus in California and Arizona. trees in many areas of southern, Epidemics of CSD in Syria have been southeastern and northern Iran (14; correlated with the presence of S. Bove, unpublished). Both N. tenellus citri-infected N. haematoceps leaf- and N. haematoceps occur in the hoppers and the host plant, S. kali. country (17). Their distribution The extremely wide distribution of throughout Iran has been well studied plants such as S. kali, which extends insofar as they are vectors of sugar- from the Atlantic to the Himalayas, . N. haematoceps favors the presence of S. citri leafhop- occurs in all citrus growing areas of per vectors over all arid and semi-arid the county. N. tenellus is less wide- areas in the warm temperature zone spread than N. haematoceps, but it where citrus is often grown, and is also is present in areas where citrus probably one of the reasons why CSD is grown. Two of the major host is present throughout much of the Old plants of the above leafhoppers are World. It has been roughly estimated well known and common in Iran: Sal- that CSD-affected trees represent sola kali and Alhagi camelorum or about 1 to 5% of all sweet orange A. persarum. Their distribution un- trees. This percentage is probably not doubtedly explains that of the two higher because N. haematoceps and Neoaliturus species. Hence, there N. tenellus are polyphagous seems to be a close correlation be- that visit citrus only rarely. tween the occurrence of N.

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