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The citrus industry is an important part of Morocco's agricultural sector and export of citrus fruit makes the third largest contribution to national export income. To improve and protect the country's citrus industry, a number of measures must be implemented by government authorities. The use of only healthy propagation material, free from debilitating pathogens, in the formation of new orchards will result in more vigorous and productive trees. Appropriate sanitary measures must be taken at the same time to prevent the invasion of mechanically or insect-transmitted viral or other pathogens into existing orchards. Recommendations are made below for actions which are of paramount importance to the prosperity of the sector.
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M., Kabbage, K., Hafidi, B. & Viennot-Bourgin, G. 1979. Présence au Maroc de Spiroplasma citri, I'agent causal de la maladie du "stubborn" des agrumes, dans les Pervenches (Vinca rosea L.) implantées en bordure d'orangeraies malades, et contamination probable du Chiendent (Cynodon daclylon L. Pers.) par le spiroplasme. C. R. Acad. Sci. Paris, 288 D: 399-402.
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Cassin, J. 1963a. Découverte de huit cas de tristeza parmi un lot de plants agés de citrus introduits au Maroc. Al-Awamia [Rabat], 9: 53-57.
Cassin, J. 1963b. Découverte de l'"infectious variegation crinkly-leaf" des citrus au Maroc. Al-Awamia [Rabat], 8: 63-75.
Cassin, J. 1964. La xyloporose (cachexiafavea) du Clémentinier au Maroc. Al-Awamia [Rabat], 10: 33-53.
Chapot, H. 1956a. Une nouvelle maladie des agrumes dans le Moyen Orient. C. R. Soc. Sci. Nat. Phys. Maroc, 22(6): 99-105.
Chapot, H. 1956b. Une nouvelle maladie à virus des agrumes dans le Moyen-Orient. Soc. Sci. Nat. et Phys. Maroc. Compt. Rend..Seances Mensuelles, 22(6): 99- 105.
Chapot, H. 1959. First studies on stubborn disease of citrus in some Mediterranean countries. In J.M. Wallace, ed. Citrus virus diseases, p. 109- 112. Berkeley, Div. Agric. Sci., Univ. Calif.
Chapot, H. 1961. Impietratura in Mediterranean countries. In Proc. 2nd Conf: IOCV, p. 177-181. Gainesville, Univ. Fla. Press.
Chapot, H. & Cassin, J. 1961. Maladies et troubles divers affectant les citrus au Maroc. Al-Awamia [Rabat], 1: 107- 129.
Chapot, H. & De Lucchi, V.L. 1964. Maladies, troubles et ravageurs des agrumes au Maroc. Rabat, INRA.
Devaux, R. 1978. Que peut-on faire contre les virus ou mycoplasmes dans une plantation? Maroc Fruits, 259: 2, 7.
Frazier, N.W. 1953. A survey of the Mediterranean region for the beet leafhopper. J. Econ. Ent., 46: 551-554.
Freitag, J.H., Frazier, N.W. & Huffaker, C.B. 1955. Crossbreeding beet leafhoppers from California and French Morocco. J. Econ. Ent., 48 (3): 341-342.
Merle, L. & Nadori, E.B. 1978. Essais de mise à fruits du Clémentier par la taille, l'incision annulaire et l'arcure. Al-Awamia [Rabat], 56: 105-126.
Nadori, E.B., Nhami, A. & Tourkmani, M. 1984. Programme d'amélioration sanitaire et de certification des agrumes au Maroc. 1st Cong. Int. Soc. of Citrus Nurserymen. Valencia, Spain.
Nadori, E.B., Quammou, M. & Quaicha, R. 1984. Etat d'avancement de la sélection nucellaire des agrumes. Journées d'étude sur les agrumes, 13- 15 mars 1984, Rabat.
Nhami, A. & Bourge, J.J. 1979. Sélection sanitaire en agrumiculture au Maroc. Al-Awamia [Rabat], 57: 29-39.
Nhami, A. & Kissi, A. 1978. Inventaires des viroses et des maladies similaires affectant le verger agrumicole marocain. Maroc Fruits, 529: 5, 12.
Nhami, A. & Zidane, A. 1984. Une contribution de la SO.DE.A.; 'amélioration sanitaire des agrumes. Maroc Fruits, 616: 2-3; 617:3.
Nhami, A., Bové, J.M., Bové, C., Monsion, M., Garnier, M., Saillard, C., Moutous, G. & Fos, A. 1980. A natural transmission of Spiroplasma citri to periwinkles in Morocco. In Proc. 8th Conf: IOCV, p. 153-162. Riverside, Univ. Calif.
Perret, J. 1953. Quelques renseignements sur les maladies à virus des agrumes existant actuellement au Maroc. Fruits et Primeurs, 23(251): 399-400.
Saglio, P., Lhospital, M., Laflèche, D., Dupont, G., Bové, J.M., Tully, J.G. & Freundt, E.A. 1973. Spiroplasma citri gen. and sp.n.: a mycoplasma-like organism associated with "stubborn" disease of citrus. Int. J. Syst. Bacteriol., 23: 191-204.
Schneider, H. 1966. South Africa's greening disease and Morocco's stubborn disease. Calif Citrog., 51(7): 299-305.
Squalli, A.H. 1978. L'agrumiculture marocaine. Bull. SASMA [Casablanca]. 23 pp.
Vogel, R. & Bové, J.M. 1964. Stem pitting sur bigaradier et sur oranger Tarocco en Corse: une maladie à virus. Fruits, 19: 264-274.
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Chapter 16: Oman
Witches' broom disease of lime nomenclature
S. citri in ornamental periwinkle in Nizwa
Virus and virus-like diseases
Defoliation and dieback of lemon and sweet lime trees at Nizwa
Horticultural practices and phytophthora Gummosis
Miscellaneous disorders, injuries and inadvisable practices
Conclusions and summary
The main citrus species grown in Oman is the small-fruited acid lime (Citrus aurantifolia) which in this chapter will simply be called lime. It is to be distinguished from the sweet (Palestine) lime (Citrus limettioides), which is also found in Oman.
Limes have been grown in Oman, and especially the northern coastal plain (Al Batinah, see Map 7), for many generations. As an export crop, having been second only to dates for a long time, by the early 1970s limes had become the most important export commodity apart from oil.
Bové carried out two surveys in Oman, one in April 1986 and the second in April 1987. Both were focused on a lethal disease, Witches' broom disease of lime (WBDL), which strikes lime trees in northern Batinah. This review also takes into account the results of a large survey undertaken, following the April 1986 survey, by the Agricultural Department of Oman to determine the extent of the disease. One of the major results of the 1986 survey was the demonstration that the agent of the disease is a mycoplasma-like organism (MLO).
In addition to WBDL in the Batinah, lime trees near Ibri and Salalah were found to be affected by another very dangerous and serious disease - citrus canker. The causal bacterium, Xanthomonas campestris pv. citri, was cultured and identified. Citrus canker was probably imported into Oman from India directly, or via the countries of the Persian Gulf.
Citrus other than lime was seen mainly at experiment stations and farms of the Ministry of Agriculture and Fisheries. Many sweet orange trees were affected by gummy bark, a disease first reported in Egypt, and many mandarin trees by cachexia-xyloporosis. Evidence of other virus and virus-like diseases, such as impietratura and exocortis, has also been found. These diseases and their agents have been introduced into Oman with budwood or trees from unreliable sources.
Witches' broom disease of lime nomenclature
The major and most characteristic symptom of the disease is the development of witches' brooms on affected lime trees, hence, the name given to the disorder in Oman. That MLOs are agents of the disease is also relevant, for it is well known that MLO-infected plants, and especially woody plants, develop witches' brooms. Hence, on the basis of symptomatology and aetiology, the name "Witches' broom disease of lime" (WBDL) has been applied. WBDL is probably one of the diseases in which the witches' brooms are most typical and well developed.
MAP 7 Oman
Oman. In April 1986, the disease was restricted to northern Batinah and extended from Al Murayr, immediately south of the border with the United Arab Emirates, to the area around Saham. Within these limits, the most severely affected areas were those of Liwa and Shinas, Al Murayr being third. According to the Omani survey of 1986, the orchards in the whole area affected had a total of 29 232 trees of which 6 291 (21.5 percent) showed symptoms of WBDL. A very similar percentage was found for individual areas of Al Murayr (21.6 percent), Shinas (22.7 percent) and Liwa (21.1 percent). The Liwa area had the highest number of affected trees (4 274) and there were approximately 185 orchards with at least one infected tree per orchard. Of these 185 orchards, 95 (51.3 percent) had I to 10 percent affected trees, 31 (16.7 percent) had 10 to 20 percent affected trees, and 15 (8.1 percent) had 90 to 100 percent affected trees. The percentage of orchards with more than 90 percent affected trees was 7 percent in both the Liwa and Shinas areas.
No affected trees were noted in the Sohar area in April 1986, but in April 1987 several orchards had trees showing symptoms of the disease. This indicates that the disease is spreading to hitherto unaffected zones in the general affected area. Until 1986; the Sohar-Saham area was the southern limit of the affected region, but by April 1987 the disease had progressed south of this border area. Witches' brooms on lime trees were seen not only in the Barka area (southern Batinah), but also inland in the Rustaq and Dhank areas. As noted above, in April 1986, the Liwa area had severely affected orchards, and in some orchards practically all trees were affected. This was the case in the orchard of Mohamed Bin Ali Al Ageeli (the Roufeli orchard) (Figs 137 to 139). The adjacent orchard belonging to Abdullah Bin Ahmed Seif Al Salami totals 100 trees, of which only five had one or two witches' brooms in April 1986. One year later, many more trees were affected, and showed well-developed witches' brooms, in spite of the fact that the farmer had already removed many witches' brooms. This indicates that when an orchard has begun to show symptoms on a few trees, progress of the disease is generally fast, a fact well known and often stated by the farmers.
In conclusion, between April 1986 and April 1987, the disease had clearly progressed within the Al Murayr-Saham area and, for the first time, trees with witches' brooms were detected outside this area at Barka, Rustaq and Dhank.
Outside Oman. The surveys carried out by the author in April 1987 indicated that WBDL was still restricted to Oman. In particular, no evidence of the disease could be found in the United Arab Emirates, at Khor Kalba, Kalba and Fujairah, immediately north of the border with Oman.
However, in 1990, clear-cut symptoms of the disease were seen in the United Arab Emirates at Hatta and Fujairah. Samples collected in the country by Taher (FAO Plant Protection Service) were found positive in Bordeaux by electron microscopy and immuno-fluorescence. These results indicate that WBDL has now reached the United Arab Emirates. In view of the rapid spread of the disease in Oman, this was to be expected.
General aspect of affected trees and progress of the disease. As already noted, affected trees are easily identified by the presence of witches' brooms (Figs 137 to 139), which are characterized by their compactness and their very small, pale green leaves (Fig. 140). These witches' brooms contrast strongly with the normal foliage of the tree (Fig. 137).
In the early stages of the disease, trees show only a small number of witches' brooms (Fig. 136), otherwise remaining totally symptomless. But the early witches' brooms are soon followed by many others in various parts of the tree (Fig. 137).
In the advanced stage of the disease, the leaves of the older witches' brooms die, but for some time the dry leaves remain attached (Figs 138 and 140). Eventually, the dead leaves fall off, leaving dead twigs and shoots as the only proof of former witches' brooms.
In the final stage of the disease, the trees have many dead twigs, shoots and branches, with only a few witches' brooms left (Fig. 139). At this stage, the trees are almost dead, and farmers are forced to eradicate them and plant banana trees instead. The progress of the disease from the first witches' broom to the last stage takes no more than four to five years.
Development of witches' brooms. A witches' broom results from the development of axillary buds that normally stay dormant. These sprout but, instead of growing into the long, normal shoots that form with normal apical growth inhibiting the development of axillary buds, they produce many secondary shoots, from which in turn other axillary buds grow. In this way, extensive ramification and internode shortening occurs. The leaves that grow on the first shoots of a future witches' broom are almost normal in size, but new leaves become progressively smaller, and new shoots become thinner as ramification continues. The thin shoots are sometimes pale green or whitish.
Hence, a well-developed witches' broom is characterized at its periphery by densely packed, thin, fragile, excessively ramified shoots with small internodes, carrying many minute leaves whose colour tends to become more and more yellow before they die and dry up.
The first witches' brooms to appear on a tree seem to develop more or less on the top or sides of the tree, but some also occur at soil level. Later, they cover the whole tree, with no clear-cut pattern of spread.
Flowers and fruits. Practically no flowers or fruits are produced on witches' brooms. Hence, fruit production falls as the number of witches' brooms increases. Bunches of flowers and fruits produced on certain shoots are much reduced in size, and quickly die. No flower virescence was observed except on periwinkles affected by WBDL (see below).
Leaves. As already noted, the many leaves at the periphery of witches' brooms are small to very small. They tend to become uniformly pale green and yellow before they eventually die and dry up. Portions of trees without witches' brooms bear normal leaves. No obvious leaf mottling seems to be associated with the disease.
Trunk branches and shoots. No particular symptoms have been observed on trunks or branches but, as already indicated above, the shoots that compose a witches' broom are excessively ramified, have short internodes and are thin and fragile. Their distal end is pale green to whitish.
The bark of shoots on witches' brooms tends to show cracks but, as the author has seen similar cracks on lime shoots in Saudi Arabia near Jeddah, in the absence of WBDL, these cracks cannot be said to be typical of WBDL.
Age of affected lime trees
Practically all the badly affected trees that the author saw were adult trees. The following observations were made in the Liwa area. In the El Ganahi orchard, the trees were 25 years old and all 140 trees were badly affected as of April 1986 - hence, the disease probably started in 1981 -82 on trees that were about 20 years old. In the El Castawi orchard, 20 affected trees were 35 years old. In another orchard, the oldest affected tree was 50 years old at the time of eradication. In the El Sotli orchard only two of about 100 trees were beginning to show symptoms of WBDL and they were 15 years old. In the Roufeli orchard, the first symptoms of WBDL also appeared in 1981 -82 on ten-year-old trees. However, in that orchard, a large witches' broom was seen on a three-year-old tree and this witches' broom had been noticed when the tree was only one year old. Electron microscopy has shown it to be infected with MLOs. In the Marhoun Dehem Al Maas orchard, three-year-old trees also showed WBDL and were pulled out.
From these observations, it seems that the disease can occur on trees of any age, although today the majority of affected trees appear to be over ten to 15 years of age. According to farmers, the disease began to strike heavily around the early 1980s. If this is the case, it must have struck trees indiscriminately, regardless of age, thus explaining why in 1986-87, four to five years later, the ages of affected trees ranged from five to 50 years. The fact that the disease shows up on young trees indicates that it does not have an incubation period of several years. Hence, the ten-, 15-, 25-, 35- or 50-year-old trees that began to show symptoms of the disease in 1981-82 were probably healthy when they were young and became infected only later. This observation supports the theory of insect vector transmission of the disease.
The WBDL MLO was graft-transmitted to one-year-old lime seedlings in Bordeaux. Symptoms appeared at 32°C within six months after inoculation, thus demonstrating that the disease can affect very young lime seedlings.
Host range of WBDL
In the field, WBDL is so far restricted to lime trees. In the Roufeli orchard, where the acid lime trees are so badly affected, sweet lime trees adjacent to badly affected acid lime trees were symptomless and no MLOs could be detected in the sweet lime tissues. In the affected areas (Al Murayr, Shinas, Liwa), no citrus species other than acid lime and sweet lime are grown. However, recent graft-inoculation experiments in the greenhouse in Bordeaux (Garnier, Zreik and Bové, 1991) have shown that Troyer citrange can be infected and is susceptible (see Fig. 141). Other citrus cultivars that have reacted are Poncirus trifoliata, Eureka lemon and rough lemon. In the United Arab Emirates, the following citrus cultivars are also affected: Palestine sweet lime, sweet limetta and citron (Gassouma, personal communication).
Identification, detection and transmission of the WBDL agent
Search for MLOs by electron microscopy. Eight samples, essentially pieces of leaf midrib, I to 2 mm long, were taken from four witches' brooms (see Table 38: samples El, E2, E4 and E7) and fixed the same day (6 April 1986) in 2 percent glutaraldehyde in 0.1 M cacody-late buffer at pH 7.4. They remained in the fixation solution until they were worked on in the laboratory at Bordeaux. Leaf samples were also taken from a multiple-sprouting formation on a lime tree (Table 38: E3), from young leaves of a sweet lime tree growing next to an acid lime tree badly affected with WBDL (Table 38: E5) and from shoots with mottled leaves on an otherwise symptomless lime tree (Table 38: E6).
Five witches' brooms (E20, E21, E22, E23 and E24) and one normal shoot from an affected tree (E25) were collected in April 1986 and carried to Bordeaux in plastic bags. Leaf samples were taken and fixed as described above. After fixation, all 17 samples were post-fixed with osmium tetroxyde, dehydrated, thin-sectioned and observed in the electron microscope. Shoots from the witches' broom were graft-inoculated on young lime seedlings.
As shown in Table 38, all witches' brooms typical of WBDL (E1, E2, E7, E21, E22, E23, E24) contained many micro-organisms in their sieve tubes (Figs 25 to 28). These organisms were surrounded by a single cell membrane, had no cell wall and were identical in all respects to the MLOs seen in other diseases of plants. There is no doubt that these organisms are mycoplasma-like. MLOs were often extremely numerous in midrib samples taken on very small leaves (5 to 20 mm in length), indeed some of the sieve tubes of such samples were literally packed with MLOs (Fig. 25). With other MLO diseases of fruit trees, such as apple proliferation, the number of MLOs is usually very low and the MLOs are not easy to find, whatever leaf material is used. In larger leaves from witches' brooms, the MLOs were less numerous and sometimes not detected at all (Table 38:E2-1, E2-2), even though smaller leaves of the same witches' brooms contained high numbers of the organisms (Table 38: E2-3,E2-4). In one witches' broom with many large leaves, MLOs were not detected in the 4-5 cm-long leaves (see Table 38: E4).
Finally, no MLOs were detected in the leaves of normal shoots from an affected tree (Table 38: E25), in the bark and small leaves of the multiple-sprouting formation found on a WBDL-affected tree (Table 38: E3-1, E3-2), in mottle-affected leaves (Table 38: E6) or in small, young leaves of a sweet lime tree growing next to a WBDL-affected acid lime tree (Table 38: E5).
These results clearly show that MLOs are associated with WBDL and that the severity of the symptoms is related to the number of MLOs in the sieve tubes - the higher the number, the smaller the leaves. It has been shown that MLOs in deciduous fruit trees overwinter in the roots and reinvade the new leaves the next spring. Thus, in spite of leaf shedding, the tree remains infected. Citrus is a non-deciduous tree, so the MLOs need not move to the roots, they are not lost by leaf shedding, and their concentration can increase continuously until they jam the sieve tubes. This is perhaps why WBDL is much more severe than MLO diseases of deciduous fruit trees, e.g. apple proliferation.
TABLE 38 Witches' broom disease of small-fruited acid lime trees in Oman: search for MLOs by electron microscopy
|Nature of witches" broom||Nature of
plant material taken on
|Detection (+)1 or no detection (-) of MLOs in sieve tube||Figure number|
|E12||Large, with small, but also normal, leaves from 3-year-old tree||Tip ends of shoots with small leaves||E1-1||+||26|
|Midribs of leaves 0.5-2cm long||E1-2||2+||28|
|E2||From tree in advanced stage of decline (Fig. 138)||Midribs of leaves 3 4 cm long||E2-1||-|
|Midribs of leaves 1 2 cm long||E2-2||-|
|Midribs of leaves 0 5-1 cm long||E2-3||4+||25|
|Midribs of leaves less than 0.5 cm long||E2-4||5+|
|E4||With mainly large normal leaves||Midrib of leaves 4 5 cm long||E4||-|
|E7||With relatively large leaves||Midribs of relatively large leaves||E7||+|
|E20||Well developed with leaves||Midribs of leaves 2 cm long about 2 cm long||E20||3+|
|E21³||Same as E20||Midribs of leaves 1 2 cm long||E21||3+|
|E22||Same as E21||Midribs of leaves about 2 cm long||E22||+|
|E23||Same as E21||E23||+|
|E24||Same as E20||Midribs of leaves about 2 cm long||E24||+|
|E25||Normal shoot from affected||Midribs of leaves about 4-5 cm tree long||E25||-|
|E3||Multiple sprouting on lime tree||Midribs of leaves less than 1 cm long||E3-1||-|
|E5||Shoots of sweet lime tree adjacent to affected acid lime tree||Midribs of young leaves 1 to 3 cm long||E5||-|
|E6||Shoots of lime tree with mottled leaves mottle||Midribs of leaves showing leaf||E6||-|
1 + to 5+: a few MLOs in sieve tubes (+) to sieve tubes crammed with MLOs (5+).
2 Samples El to E7 were fixed in Oman.
3 Samples E21 in E25 were fixed in Bordeaux
Colour test for MLOs of WBDL. A simple but non-specific cytochemical technique has been described for the detection of MLOs in the sieve tubes. It uses a special stain (DAPI) that combines specifically with DNA (Russel, Newman and Williamson, 1975). The stain (DAPI: 4.6 diamidino-2-phenylindole 2 HCl) is available commercially from Germany. This technique was thought to be particularly applicable in the case of WBDL, since so many MLOs (and hence DNA) are present in the sieve tubes of leaves from witches' brooms.
Leaf samples were collected from the witches' brooms taken to Bordeaux (E20 to E24). Leaf midribs were fixed for two hours in 5 percent glutaraldehyde in 0.1 M phosphate buffer at pH 7.4. Sections of 30 µm thickness were obtained with a freezing microtome. The sections were immediately transferred to the DAPI solution (1 µg/ml) for ten minutes, mounted on a microscope slide and sealed with wax or varnish. They were observed in a fluorescence microscope equipped with a high-pressure mercury lamp, 365 µm excitation filter and 450 µm emission filter.
The samples from witches' brooms showed a high DAPI fluorescence in the sieve tubes, while no fluorescence was seen in sieve tubes from healthy lime leaves. These results show that DAPI coloration can be useful in work with WBDL.
Attempts to culture the MLO associated with WBDL. Some of samples E20 to E24 were used to try to culture the MLO associated with WBDL. The technique was that used with Spiroplasma citri-infected citrus material (Bové, Whitcomb and McCoy, 1983).
Two of the best media were used: MIA and SP4 (Whitcomb, 1983), but the MLO of WBDL could not be cultured, although several attempts were made to do so. This result is not surprising since, at the present time, no MLO has been obtained in culture. The only culturable phytopathogenic mycoplasmas are the helical spiroplasmas.
Transmission of the WBDL MLO. The WBDL MLO was graft-transmitted to young lime seedlings. Symptoms on inoculated plants are identical to those seen in the field, and require high temperature conditions (32°C rather than 25°C) for expression. Graft transmission to
Troyer citrange (see Fig. 141), Eureka lemon, rough lemon and P. trifoliata was also achieved. In addition, the MLO was transmitted from symptomatic lime seedlings to periwinkle (Catharanthus roseus) plants by dodder (Cuscuta campestris) (see Figs 29 and 30). Symptoms on periwinkle plants are similar to those on lime - witches' brooms with very small leaves (see Fig. 142). Flowers are small and show virescence. Back transmission from periwinkle to lime has also been achieved.
Monoclonal antibodies and DNA probes for detection of the WBDL MLO WBDL MLO-infected periwinkle leaves have been used to prepare monoclonal antibodies (MAs) and DNA probes against the WBDL MLO. Eighteen such MAs and five DNA probes have been obtained.
These reagents make it easy to detect the MLO in plant material (see Fig. 143) and putative insect vectors (Garnier, Zreik and Bové, 1991). The use of the DNA probes has shown that the WBDL MLO shares DNA sequences with the MLO of sesamum phyllody.
WBDL as an MLO disease. As already indicated, MLOs are clearly involved in WBDL. They are very probably the causal agent of the disease, but absolute proof of this must await fulfilment of Koch's postulates. As long as the MLOs are not available in culture, these postulates cannot be fulfilled and, to be exact, the MLOs can only be said to be associated with the disease, not its causal agent. It is with these restrictions in mind that the expression "MLO disease" can be used.
Spread of WBDL
From discussions with Mohamed El Sheshtawi, Rashwan Mohamed Rashwan and several farmers, it appears that the disease was first noticed around 1975, but that the major damage has occurred since the early 1980s. The Roufeli orchard, so badly affected in 1986 (Figs 137 to 139) was still in good shape in 1981-82. In the El Ganahi orchard, which has 100 percent affected trees at the time of writing, the disease followed a similar evolution. The progression of the disease from April 1986 to April 1987 was dramatic in certain orchards. Areas free of WBDL in 1986 showed symptoms of the disease in 1987, trees that were symptomless in 1986 had symptoms in April 1987. The urgent question now is - do these observations mean that WBDL is spreading from tree to tree, from orchard to orchard, from northern Batinah to southern Batinah, from Oman to the United Arab Emirates? If so, is it caused by propagation of infected plant material, or natural transmission by insect vectors, or both?
Marcottage is the main propagation technique for lime trees in Oman. Farmers do not use seedling trees, cuttings or budlings. It is, however, very difficult to obtain reliable information from the farmers as to where and from whom they obtained the planting material of the old lime orchards which are affected by WBDL today. They do not keep records, and are not aware of their importance. Hence, it is not known whether badly affected orchards were propagated from healthy or infected trees and so the role of infected planting material in the spread of the disease cannot at present be assessed.
However, for the following reasons, the use of infected plant material does not seem to be the major cause of the explosive way the disease appears to have spread since 1981 -82. As indicated above, trees of all ages show the disease. The fact that young trees are affected shows that the incubation period - the period it takes for an infected tree to show symptoms -is short, not longer than several years. This is confirmed by the graft-transmission experiments mentioned above which show that young, one-year-old lime seedlings show symptoms within six months of graft inoculation. Hence, the ten-, 15-, 25-, 35- or 50-year-old trees that began to show symptoms around 1981-82 had become infected only a relatively short time before - they were not infected in their early years, or they would have shown symptoms long ago. This logically means that they must have become infected through vector transmission of the disease agent, a conclusion in keeping with the MLO nature of WBDL. It is well known that MLO diseases of plants are naturally transmitted by insect vectors, essentially leafhoppers, but also psyllas (in the case of pear decline, for instance), so it would not be surprising if WBDL were also transmitted in this way. the many witches' brooms, with their numerous small leaves so rich in MLOs, would be efficient sources of inoculum for the vectors, but infected plants other than lime should a]so be considered as potential sources of contamination. Natural transmission of WBDL and identification of the vector are priority research projects. The recently developed reagents to detect the WBDL MLO (MAs and DNA probes) should make it possible to identify the vector(s) quickly. Table 39 lists leafhoppers that have been reported in Oman.
Regarding natural transmission of WBDL, the fact that badly affected orchards (Roufeli, El Ganahi) exist alongside orchards with only very few affected trees suggests tree-to-tree propagation.
TABLE 39 Leafhoppers identified in Oman
|Common name||Scientific name||Locality||Host|
|Leafhopper||Amrasca devastans||Northern Oman||Alfalfa|
|Cotton jassids||Amrasca devastans||Brinjal/okra|
During a May 1991 survey in Oman for potential leafhopper vectors of the WBDL MLO, the Bordeaux group in collaboration with Omani colleagues, and especially A.M. Mjeni, found the leafhopper Hishimonus phycitis to be consistently associated with lime trees, both healthy and affected. This was the first time this leafhopper was reported in Oman and the Arabian Peninsula. In a February 1993 survey, the same leafhopper was also found on lime trees in the United Arab Emirates (Al Shareqi, Gassouma, Garnier and Bové, unpublished). H. phycitis is well known in India and Pakistan where it is recognized as the vector of the MLO of little leaf disease of eggplant. Experiments are under way to assess the role of H. phycitis in the transmission of the WBDL MLO.
Finally, the spread of WBDL to the United Arab Emirates has now occurred, as noted above. The disease was seen for the first time in 1989. Acid lime samples collected by Taher (FAO Plant Protection Service) and analysed in Bordeaux were found to contain the
WBDL MLO when examined by electron microscopy and immunofluorescence. Leaf samples of citron, Palestine sweet lime and sweet limetta from the United Arab Emirates sent to Bordeaux by Gassouma were found positive by some techniques.
Origin of WBDL
The origin of WBDL is unknown. No information was obtained in talks with farmers or extension officers. WBDL is a new disease, described for the first time in Oman. Because of its severity, if it had occurred elsewhere, it would probably have been noticed. This is precisely what has happened in the United Arab Emirates where the disease was suspected for the first time in 1989. No symptoms of WBDL could be seen in the Islamic Republic of Iran in 1985, in Pakistan in 1987, or in India in 1988 and 1990. In India citrus is affected by another MLO disease- rubbery wood (Alhawat and Chenulu, 1985). The symptoms and host range of WBDL and rubbery wood are clearly different, and the two diseases do not seem to be related. In the light of the fact that S. citri, the agent of stubborn disease of citrus, can infect many plants other than citrus (for instance, S. citri-infected periwinkles were discovered at Nizwa), it is not unlikely, but in no way proved, that the MLO of WBDL could come from a plant other than citrus with the involvement of an insect vector. In this respect, sapotilla trees affected by fasciation have been hypothetically related to WBDL. However, the cause of fasciation is not known; it could be varietal. Sapotilla trees with fasciation occur at the Rumais Research Centre and in the Salalah region.
Attempts to control WBDL
Farmers have cut off witches' brooms in the hope of eliminating the disease, but in all cases they have reappeared. This observation is in total agreement with the infectious, MLO nature of the disease. It also shows that MLOs are present, not only in the witches' brooms, but also in asymptomatic parts of the trees, such as large branches. When such branches produce shoots, the MLOs are able to invade the developing shoots and produce a witches' broom. Thus, MLOs infect WBDL-affected trees in a systemic way. Making marcotts from symptomless shoots of affected trees will undoubtedly result in MLO-infected progeny trees. Therefore, WBDL-affected trees, and even symptomless trees in WBDL-affected orchards, should not be used as parent trees for propagation purposes.
Some attempts to control WBDL by tetracycline injections into affected trees have been made at the orchard of Salem Hamdan Al Amri at Shinas. However, these injections were not carried out in a proper way and so gave no results. In addition, the multiple "trunks" characteristic of so many lime trees in the Batinah make efficient injections difficult. Such trees require at least as many injections as there are "trunks" while, in the case of trees with single trunks, two injections per tree would probably be enough.
Finally, because of the severity and destructive nature of WBDL, farmers have begun to pull out whole orchards and replace citrus with other crops such as banana.
Orchards with a high percentage (50 percent or more) of badly affected trees (for instance, Salem Hamdan Al Amri orchard in the Shinas area; Roufeli orchard and Al Salami orchards in the Liwa area) should be totally uprooted and burnt, indeed, farmers have already begun to do this. It does not pay to keep a few symptomless trees when most trees in the orchard are affected and have to be pulled out. In less severely affected orchards, all trees showing symptoms should be uprooted and burnt. By removing the infected trees, one removes the source of inoculum on which the probable insect vectors become contaminated, thus slowing the spread of the disease. Serious consideration should be given to taking even more drastic measures and destroying all affected orchards - in other words, working towards the complete eradication of the disease.
Three orchards, each with at least 100 trees' should be preserved for experimentation purposes: one badly affected orchard (75 percent affected trees or more), one less affected orchard (25 percent affected trees) and one mildly affected orchard (10 percent affected trees). These orchards should be used to investigate: the distribution of MLOs in a ten-year-old lime tree showing only one single witches' broom; whether seedling lime trees and seedlings of other citrus species become infected, and how quickly, when exposed to natural contamination within one of the three orchards, among affected or symptomless trees; and whether seedling trees are protected from infection if placed in an insect-proof cage. The MAs and DNA probes now available are indispensable for this work
Apparently healthy orchards in which affected trees have been pulled out will have to be surveyed at least twice a year to determine if symptoms have reappeared. Trees on which symptoms do appear must be marked, so as to follow the eventual progress of the disease in the orchard.
Movement of citrus material out of the Al Murayr-AI Khaburah area must be strictly prohibited, in order to prevent the contamination of areas still free of WBDL. It would be a very bad mistake to take young trees from the affected area into areas as yet free of the disease. These regions should be regularly surveyed to assess absence of WBDL.
As stated above, lime trees should not be propagated from trees - symptomless or otherwise - in infected orchards, as lack of symptoms is no guarantee that the trees are not infected. Nursery workers should be instructed accordingly. The production of lime trees from seed rather than marcotts should be considered. Citrus nurseries in the affected areas must be carefully surveyed for WBDL.
The existence and spread of WBDL should be notified and not kept more or less secret. On the contrary, citrus growers and extension officers should receive full information about the disease and the severe threat that it represents for the lime industry, not only in Oman but also in other countries. Public meetings and television broadcasts could be used to good effect to increase awareness of the problem.
Quarantine measures should be reinforced in order to prevent illegal importation of citrus material from countries such as Pakistan and India where tristeza, greening and citrus canker exist. Citrus material should only be introduced from sources such as France, Spain or the United States of America which can certify that their citrus plant material is free of infectious agents.
The facilities required for, and the financial implications of, the research programme are outside the scope of this review. Yet, a large insect-proof screenhouse is a prerequisite for transmission studies. For safety reasons, these studies should be carried out within, or close to, the affected Shinas-Liwa area, not at Rumais.
WBDL, limited to Oman until 1989, but now also present in the United Arab Emirates, is a new infectious MLO disease of citrus, very probably transmitted by insect vectors. the danger of WBDL, not only for Oman, but also for other countries, cannot be overemphasized. It is one of the most severe diseases of citrus, very much more severe than, for instance, stubborn disease, another mycoplasma disease of citrus. The destructive potential of WBDL will be even greater if it turns out that the disease can affect citrus species other than acid lime. It has already been shown that Troyer citrange is susceptible.
The disease seems to have appeared in the late 1970s, and it began to spread rapidly in the early 1980s. It affects trees of any age. It is not known how and why the WBDL started.
Was infected plant material imported from abroad into Oman, with or without a vector? Was infectious plant material present in Oman but without a vector and the vector, possibly H. phycitis, introduced only recently? These questions and others are important not only for academic reasons but also for disease control. Only research will provide the answers.
Recommendations have been made concerning limitation of the spread of WBDL. It would even be advisable to try to achieve total eradication of the disease, if this is in any way feasible, though now that the disease appears to have spread to the United Arab Emirates this will be extremely difficult.
All those involved in the citrus industry in Oman must be made aware of the seriousness of the situation created by WBDL, and of the threat that the disease represents, not only for Oman but also for other citrus-growing countries. All efforts should be made to study and control the disease.
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