Stubborn in the Syrian Arab republic

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The Latakia-Tartus coastal area

Map 3 on p. 71 is of western Syria. Table 17 shows that stubborn is widespread throughout the Latakia-Tartus citrus-growing area, with practically all mature Washington navel or Valencia late sweet orange orchards having at least some stubborn-affected trees.

In one orchard (Sidawi orchard at El Annadeh, 20 km south of Latakia) stubborn in Washington navel sweet orange trees was very severe. The owner had top-grafted the trees with Jaffa sweet orange. Table 16 shows that the Jaffa part of all top-grafted trees became infected with S. citri through the underlying Washington navel scion.

As shown in Table 17, stubborn disease mainly affects Washington navel (Figs 148 to 150, 167 and 285) and Valencia sweet orange trees, these being the main varieties grown. However, Hamlin, Jaffa, Butler and Navelina sweet orange trees as well as Ortanique tangor and shaddock have also been found to be affected, though less severely.

In 1986, the Agricultural Services in Latakia determined the percentage of stubborn-affected trees in several orchards planted in the period 1979-81. Table 18 shows that, in general, the percentage of affected trees is highest in orchards located close to the Mediterranean coast, especially when the trees were planted in 1979-80 rather than in 1981. Table 18 shows that these observations are particularly true for the 1979 orchards of the FAO project in El Annadeh and Amrit.

The FAO project

As part of a UNDP/FAO integrated Agricultural Development Project, a citrus nursery and several mother blocks were established from 1978 at El Annadeh. Mother blocks were also planted at Amrit near Tartus. Both locations are close to the Mediterranean coast. The budwood, certified free of known diseases, was introduced from the Citrus Experiment Station in Corsica between 1978 and 1981. In 1982, many trees with stubborn symptoms in the mother blocks were observed and these trees were shown to be infected with S. citri. An FAO Technical Cooperation Programme project was then started in order to try to elucidate the reasons for this high rate of infection. At the end of three years of investigation, it was observed that the percentage of stubborn-affected trees was 33 percent in the sweet orange mother block (see Table 19; Figs 285 and 286) and 45 percent in the grapefruit mother block (see Table 20; Figs 287 and 288). Thirty-three out of 34 sweet orange varieties and all eight grapefruit varieties showed symptoms of stubborn, but only a few mandarins showed infection (Fig. 289). Similar observations were made at Amrit (see Table 21).

Two explanations could be offered to account for the high incidence of stubborn. Either the budwood received in Syria was already infected with S. citri in Corsica or it was free of S. citri, but the trees propagated from it became infected in Syria through natural transmission of stubborn. Both hypotheses were investigated.

Evaluation of budwood from Corsica

The citrus budwood shipped to Syria was taken from trees in the mother block of the San Giuliano Citrus Experiment Station. These trees had been indexed free of virus and virus-like diseases by Vogel prior to 1978. Indexing for stubborn disease had been carried out in a heated greenhouse at 32°C and repeated several times, always with negative results. Hence, the budwood was considered to be free of S. citri.

TABLE 17 Spiroplasma citridetection by ELISA and culture in adult citrus orchards in the Syrian

TABLE 18 Percentage of stubborn - affected trees in various orchards in the Latakia Tartus region

When, in 1982, it was found that many trees propagated from the Corsican budwood were infected with S. citri in Syria, fruit from the same mother trees from which budwood for Syria had been taken was collected and sent to Bordeaux for analysis. There, the fruit was analysed for the presence of S. citri by ELISA and by culture. The results of these tests, carried out in January 1983, were all negative. The same tests were repeated in the winter of 1983/84, again with negative results. In early October 1984, a search was conducted in Corsica in an attempt to detect S. citri in citrus leaf material and in experimental periwinkles, by ELISA and by culture. All these assays were negative. Finally, the citrus trees in the mother block at San Giuliano were carefully examined for stubborn symptoms, but none were seen.

All these negative results can be explained in two ways: either S. citri is indeed absent and the Corsican trees are truly free of it, or the trees are infected, but the pathogen is present in such a low concentration that it escapes detection by ELISA and by culturing. The latter possibility must be considered for two reasons. First, the optimum temperature for the growth of S. citri is around 32°C. When infected trees grow at this temperature, S. citri multiplies well in the tree and reaches a concentration at which symptoms develop, and the organisms can be detected by ELISA and by culture. In Corsica, the temperatures are much lower than 32°C, even at the height of summer. Expression of stubborn symptoms therefore would be poor or not occur. In this case, S. citri detection gives false negative results. When, however, the same plant material is placed at temperatures favourable for S. citri multiplication, symptoms will eventually occur. It should be remembered, however, that, in Corsica, when indexing for stubborn at 32°C in the greenhouse, no symptoms had appeared. Second, the leafhopper N. haematoceps, now known to be a vector of S. citri, is present in Corsica (Brun et al., 1988), and the insect is occasionally found to be infected with S. citri.

For these reasons, the health status of citrus budwood in Corsica had to be further assessed. This was done by reindexing budwood derived from the mother block in San Giuliano for stubborn at 32°C. All the tests were negative.

A complementary test was also started at El Annadeh in Syria. Five hundred buds of Washington navel sweet orange (clones 39, 141, 204, 205 and 215), known to be infected with S. citri in Syria (see Table 19), were collected in Corsica and used for budding in situ under screenhouse conditions (Fig. 290) and on rootstocks treated with the systemic insecticide Temic. None of the screenhouse-grown plants developed symptoms of stubborn.

These assays and experiments indicate that the budwood from Corsica was free from S. citri. Therefore, only natural transmission of S. citri remains to explain the high incidence of stubborn in the FAO project, as well as in other orchards that were established in 1979-8O, such as those at Hmaymin (No. 1 in Table 18) or Saleh-Basatine (No. 6 in Table 18).

Natural transmission of S. Citri in the Syrian Arab republic

Several approaches were followed to study natural spread of S. citri in Syria.

The leafhopper N. haematoceps found infected with S. citri

Leafhopper collections were made at random with a D-vac aspirator (Fig. 291) in citrus orchards and on other plants. Forty-one species of leafhopper were identified (see Table 22), of which 21 were analysed for S. citri by ELISA and by culture. Each batch consisted of from five to ten leafhoppers. A positive reaction implied that at least one out of the batch of insects was infected. Only one species of leafhopper, N. haematoceps (see Fig. 34), was found to be infected with S. citri. This is the same species that had been found to be infected in Morocco in 1979 and, subsequently, in Corsica in 1984, Iraq in 1986 and Cyprus in 1987.

N. haematoceps is closely related to N. tenellus, the sugar beet leafhopper in California and Arizona, but the two species can easily be separated by the anatomy of their male plates. Regarding their vector properties, N. haematoceps is regarded as the most important vector of curly top virus of sugar beet in Turkey (Bennett and Tanrisever, 1958), as is N. tenellus in California. Both species are vectors of S. citri-N. tenellus in the United States of America (Oldfield et al., 1976) and N. haematoceps in the Mediterranean (see below). The two species are also thought to be similar in general habitat (Nielson, 1968). High populations of N. tenellus are commonly found in warm or hot, arid or semi-arid regions. Studies on the influence of temperature on the duration of egg and

TABLE 19 Stubborn-affected trees in the sweet-orange mother block of the El Annadeh project

Notes:
¹ Trees removed in November 1982 as a result of positive S. citri detection in October 1982.
² As of October 1984.

In Syria, N. haematoceps was found only in low numbers in citrus orchards and on cultivated plants in the coastal area from Latakia to Tartus. However, larger numbers were collected in the dry, sandy, non-cultivated areas adjacent to the El Annadeh project, such as Fidio, as well as in many other sites along the coast (see Map 3). Exceptionally high numbers of N. haematoceps were collected in the Ghab (Orontes) Valley on a few stock plants (Matthiola annua or Cheiranthus matthiola) growing as ornamentals in the garden of the Apamea Mosaic Museum. S. citri was cultured from all samples analysed (five leafhoppers per sample). (It should be noted that, in Corsica, N. haematoceps could also be collected on wild stock plants growing on the dry, sandy east coast.) N. haematoceps was also captured in the ruins of Palmyra, where it was also found to be infected with S. citri. The fact that N. haematoceps infected with S. citri could be collected both in the Ghab Valley near Apamea and other sites (see Map 3) and in Palmyra, far from the citrus-growing coastal zone, indicated that there must be host plants other than citrus that harbour this pathogen. This is an important conclusion in trying to explain natural transmission of S. citri in Syria.

TABLE 20 Stubborn-affected trees in the grapefruit mother block of the El Annadeh project

Notes:
¹ Trees removed in November 1982 as a result of positive S. citri detection in October 1982.
² As of October 1984.

Salsola kali, the preferred host plant of N. haematoceps

As indicated above, high numbers of N. haematoceps leafhoppers were collected in the arid, non-cultivated areas surrounding the El Annadeh site of the FAO project as well as in many other sites along the coast (see Map 3). The plant on which these leafhoppers were captured was identified as S. kali (see Figs 36 and 170 to 174), a Chenopodiaceae.

This plant is the preferred host of N. haematoceps. It is also the major host of N. tenellus in the United States of America where it is called Russian thistle or tumbleweed. S. kali is very widespread, with its natural distribution area covering the Mediterranean zone and most of the Irano-Turanian regions. In Cyprus, Iran, Iraq, Morocco and Syria the species is extremely common, particularly on disturbed habitats (ruderal sites in towns, villages, on roadsides, etc.) in arable land and on the seashore (see Map 3 and Figs 170 and 171). It grows at altitudes of 2 000 m and higher, but its highest densities are reached in the hot and arid to semi-arid lowlands. In Syria, S. kali is extremely frequent along the coast and its distribution explains the distribution of N. haematoceps (see Map 3 and Fig. 170). The leafhopper has been captured on S. kali at numerous sites all along the coast from Amrit, south of Tartus, up to Latakia, and many of the N. haematoceps samples were infected with S. citri (Fig. 173). This also explains why the incidence of stubborn is higher in orchards close to the coast, where S. kali and N. haematoceps are much more abundant (see Table 17).

TABLE 21 Stubborn-affected trees in the mother block at Amrit

The FAO citrus projects at El Annadeh and Amrit are close to the coast. In 1986 S. kali was seen growing as a weed in between the rows of a new extension of the citrus nursery at El Annadeh (Fig. 174). Undoubtedly, the same situation must have occurred in 1978-80 when the project was first started. The presence of S. kali within the newly planted mother tree blocks must have favoured an abundant population of N. haematoceps and, hence, a high incidence of S. citri transmission to the young trees. This explanation assumes that N. haematoceps is indeed a vector of S. citri. However, the mere fact that the leafhopper is found to be infected with S. citri does not suffice to make it a vector. Experimental transmission of S. citri by the leafhopper must be demonstrated.

Experimental transmission of S. citri by N. haematoceps

N. haematoceps leafhoppers free from S. citri were received from Corsica in July 1984 and raised in Bordeaux. Third generation individuals were injected in November 1984 with a pure culture of S. citri, obtained from infected N. haematoceps collected in Palmyra in October 1984. The injected leafhoppers supported the multiplication of S. citri to high titres (10⁷ colony-forming units/ml) and were able to transmit the spiroplasma to healthy periwinkle plants. These plants developed leaf yellows symptoms, and S. citri was later reisolated from them. In addition, N. haematoceps leafhoppers were able to acquire S. citri by feeding on infected periwinkle plants and to transmit it to healthy plants. Transmission could be easily achieved with one infected leafhopper per plant. These experiments proved that N. haematoceps is a vector of S. citri (Fos et al, 1986).

Natural transmission of S. citri to periwinkle

Periwinkles exposed to natural infection in various locations from north of Latakia to south of Tartus became infected with various MLOs. Sixteen periwinkle plants became contaminated with a hitherto unknown spiroplasma, Spiroplasma phoeniceum (Sail lard et al., 1987). Six plants showed infection by S. citri (see Table 23; Fig. 292).

The number of periwinkle plants that became infected with S. citri in these experiments was low, indicating a low incidence of 5. citri transmission. The reason for this is now clear. When these experiments were initiated very little was known about natural transmission. The periwinkle plants were established within adult orchards containing a high number of stubborn-affected trees. Today it is know that natural transmission of S. citri within such orchards is low. The incidence is much higher in young, one- to three-year-old orchards and even higher in non-cultivated areas, close to the coast where S. kali is abundant. In 1985 in one such location (Fidio site), close to the El Annadeh project, several periwinkle plants became infected with S. citri.

TABLE 22 Relative frequency of leafhoppers and planthoppers in the Syrian Arab Republic and number of samples analysed for presence of Spiroplasma citri

TABLE 23 Periwinkle (Catharanthus roseus)plants naturally infected with Spiroplasma citri or S. phoeniceum in the Syrian Arab Republic

The search for infected seedling trees

5. citri does not pass through the seeds of affected fruits. Hence, seedling trees are free of stubborn. As is known, nucellar trees are seedling trees resulting from the development of a nucellar embryo. Nucellar trees are therefore free of S. citri unless they become contaminated by insect vectors.

Two of the seven Orlando tangelo seedling trees in the plot devoted to citrus seed production at the El Annadeh project were found to be infected by S. citri. Nucellar trees of Hamlin sweet orange had been obtained at the fable Experiment Station. Young trees propagated from this nucellar Hamlin were grown in one of the nurseries at El Annadeh. One of these trees was also infected with S. citri.

Finally, three out of six Madame Vinous sweet orange trees propagated at El Annadeh from budwood of a tree always kept in an insect-proof screenhouse in Corsica showed S. citri infection.

Conclusion

At the end of several years of research on stubborn and its transmission in Syria, a general picture emerges that satisfactorily explains the high incidence of stubborn observed in some of the orchards that were planted in 1979-80.

The vector, or at least the principal vector of S. citri, has been identified as the leafhopper IV. haematoceps. This leafhopper was frequently found infected with S. citri in nature. S. kali is the preferred host plant of the leafhopper and, since this plant is widespread along the Mediterranean coast of Syria, the stubborn vector is also abundant there. When new orchards of Washington navel and Valencia late sweet orange trees are planted close to the coast on reclaimed land, the presence of S. kali as a weed within or around the young orchards attracts the stubborn leafhopper and transmission eventually occurs. This is what has very probably happened at the El Annadeh and Amrit projects, and at private orchards such as those located close to the coast at Hmaymin or Saleh-Basatine (see Table 18). At El Annadeh, the incidence of stubborn is higher in the orchards planted in 1979 and 1980 than in those of 1981. This might be due to the fact that, by 1981, three to four years had elapsed since the land was initially reclaimed. The population of S. kali had probably decreased during that period as a result of horticultural practices, weed control, etc. It is, however, not excluded that the population dynamics of IV. haematoceps are controlled by as yet unknown factors other than the presence of S. kali, and that epidemics of stubborn occur only in certain years, such as 1979-80 in Syria.

In California, it is a well-known fact that epidemics of stubborn occur only in certain years. During those years, a high incidence of S. citri transmission is observed. For instance, 1973 was an epidemic year in California. In one location (Moreno), 76 out of 105 plants became contaminated, while in the following years very little transmission took place.

In the Mediterranean area, stubborn disease was first reported in the country then known as Palestine in 1931 by Reichert and Perlberger, who indicated that the disease was in an epidemic form and was causing serious damage to young citrus orchards. More recently, in the Ghor (Jordan River valley), young citrus plantings established in a non-citrus-growing area showed a high incidence of stubborn infection. A similar situation might have occurred when the El Annadeh project was started. The land was cleared in 1978 and the natural vegetation removed. This left naked, sandy ground, with no windbreaks around the plots to be planted with citrus. The first trees in this open land were those in the sweet orange and grapefruit blocks. Without much natural vegetation except for S. kali plants, these young trees probably became the alternative host plants on which leafhoppers could feed. In other words, leafhoppers were practically forced to invade these young orchards. Part of the land of the El Annadeh project has not yet been cleared or cultivated (Fidio area). Interestingly enough, N. haematoceps was captured in 1984 in this undisturbed area. This situation resembles that of El Annadeh before it was cleared in 1978. In short, it is believed that land-clearing operations under semi-desert conditions favoured leafhopper migration and feeding on young citrus trees, resulting in a local epidemic. It is, of course, the presence of S. kali in the area that accounts for the presence of the leafhopper vector of stubborn.

Besides such a localized increase in vector activity in a limited area, climatic and other factors could favour a general increase in N. haematoceps, resulting in more generalized epidemics in certain years. The understanding of such occasional epidemics requires further study of the biology and ecology of N. haematoceps. In particular, it is important that herbaceous host plants naturally infected with S. citri be identified in Syria. Finally, another important epidemiological consideration is that young citrus trees are especially susceptible to stubborn infection.

In California, nursery trees and trees in young orchards have been observed to be the main target for infection. Once these trees reach the age of six to seven years, their chances of becoming infected are low. This is very probably due to the fact that N. tenellus in California and N. haematoceps in Syria are not citrus leafhoppers. Their encounter with citrus is only occasional. The younger and smaller the trees, the higher the chances that these insects can penetrate deeply into the orchards and feed on them. At the El Annadeh project, the sweet orange and grapefruit mother trees that became infected were only two years old when they showed stubborn symptoms.

More work has been carried out on stubborn in Syria than anywhere else in the Mediterranean region. However, there is still insufficient knowledge about the biology and ecology of N. haematoceps, and about the herbaceous plants possibly infected with S. citri in Syria. This information is needed to explain stubborn epidemics better in this and other countries.

In the meantime, the following recommendations are given to assist in keeping stubborn disease under relative control.

• Use only budwood free of virus and virus-like pathogens.
• Since stubborn is an insect-transmitted disease, nurseries should be established in areas free of S. kali, where the incidence of stubborn is very low and where only very little or no transmission of S. citri occurs.
• If no suitable areas for nurseries can be found, nursery trees must be grown in greenhouses or screenhouses (Fig. 290). If this is not possible, young trees in open nurseries should be protected with a systemic insecticide (dimethoate, Temic).
• If stubborn is a problem, citrus varieties more tolerant than navel oranges should be used. They should not be top-grafted to stubborn-affected trees.
• Nurseries and young orchards should be surveyed at least once a year, and all infected or suspect trees should be pulled out and replaced by healthy ones.

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