Citrus Greening and Psylla Vectors of the Disease in the Arabian Peninsula J. M. %vC and M. Garnier ABSTRACT. Greening disease of citrus was discovered in the Arabian Peninsula in two surveys of virus and virus-like diseases for FAO. Identification of the disease was based on symptom expression and on electron microscopical detection of the green- ing organism. The disease was found in Saudi Arabia in the areas of Taif, Turabah, Korhma and Bisha. The insect vector, Diaphorina citri, reaches extremely high popu- lation levels at certain times of the year (November). The disease is very probably re- sponsible for the decline and disappearance of sweet orange and mandarin trees in the affected areas. Today, Mexican lime is the only citrus species still grown. Since Mexican lime is relatively tolerant of greening and is also an excellent host for D. citri, efficient transmission of the greening organism is favored. The type of greening involved is the heat tolerant, Asian one, a more severe form of the disease. Greening is also present in North Yemen. It occurs only at elevations above 1000 m or so (Taiz, Hamman Ali). It is not present along the coast. The vector is Trioxa erytreae and the type of greening involved is the African one, a less severe form. The presence of the two forms of greening and the two psylla vectors along the East coast of the Red Sea is a threat to the citrus growing areas of the Arabian Peninsula and to the citrus industry of the Mediterranean Basin and the Near East. Index words. citrus, greening, psylla, Saudi Arabia, Yemen. "Greening" is one of the most when plants grow at temperatures serious diseases of citrus and exists above 32°C. No GOs are found in on the African continent south of such symptomless trees (3) and the the Sahara in citrus growing African strain of the GO is prob- countries including Ethiopia. The ably unable to multiply at such disease is not present in the north- temperatures. The heat sensitivity ern parts of Africa. It is trans- of the African GO and the African mitted in Africa by the "African" psylla vector probably explains the psylla, Trioxa erytreae (Del geographical distribution of green- Guercio) (11). This vector is sensi- ing disease in Africa. tive to high temperatures and low A second form of greening is humidity, and it is often confined the Asian greening. It is a more to cool, elevated areas, as is the severe and more widely distributed disease. For instance, greening form of the disease. Affected citrus affects the "highveld" areas of areas extend from China and Transvaal and Swaziland but not southeast Asia to Nepal, India and the "lowveld" regions of Swaziland. Pakistan. In Asia, the GO is trans- Similarly, in the island of Madagas- mitted by the Asian psylla, Diac car, greening and T. erytreae are phorina citri (Kuwayama) (5, 6, only present on the high plateau, 9) but we have shown that the not on the coastal areas. The same African psylla can also transmit is true in Kenya where greening is the Asian strains of the GO (10). severe in the citrus areas above The Asian psylla is a much sturdier 700 m but absent from the coastal insect than T. erytreae. It resists regions. A Gram-negative bacteri- high temperatures and low humidi- um-like organism is present in the ties. Similarly, the Asian strain of sieve tube elements of affected the GO is heat tolerant (3). Hence, plants (4). This "greening organ- the geographical distribution of ism" (GO) is thought to be the Asian greening occurs not only in causal agent of the disease. The relatively cool areas, but also in GO of African greening is sensitive hot coastal zones. to heat, and no symptoms develop The two forms of greening as Ninth IOCV Conference well as the two psylla species occur in a Siemens Elmiscope 101 elec- together on Reunion island and tron microscope. Mauritius island. In Reunion, D. RESULTS AND DISCUSSION citri is present from sea level up to an elevation of 600 m, and T. ery- Asian form of greening disease treae only above 600 m. Greening and D. citri in Saudi Arabia. Clear- disease extends from sea-level up cut symptoms of greening disease to 1000 m. were observed on Mexican lime Greening is not present in the trees in the following plateau oases countries surrounding the Mediter- in the southwestern part of the ranean sea, nor does it affect country (fig. 1) : Zaymah, Taif Jordan, Iraq and Iran. However in (1682 m), Turabah (1450 m), November 1981 and April 1982, Khurmah, Bisha. An organism while conducting a survey for virus identical to the greening organism and virus-like diseases of citrus on (GO) as known elsewhere was de- behalf of FAO, J. M. Bov6 dis- tected by electron microscopy covered greening disease in Saudi (EM) in affected citrus material Arabia and North Yemen. from all these areas (fig. 2 A, B, C). These EM results confirm that the MATERIALS AND METHODS symptoms observed are those of Diagnosis of greening was greening. based on symptom expression and In the above areas, the only electron microscopical detection of citrus species now grown is Mexi- the GOs in the sieve tubes of leaves can lime. According to citrus grow- and/or fruits. ers, sweet orange and mandarin Leaves and fruits were kept in trees used to do well, but have plastic bags for a few hours until perished within the last ten years. they could be further processed. Greening disease is very probably The leaf midrib was cut out with the cause of this destruction since a razor blade and chopped into 2 to severe symptoms of greening were 4 mm long pieces. About five such observed in diseased sweet orange pieces were fixed by immersion into and mandarin trees at Taif, Tura- a 5 ml screwcap tube filled with 2% bah and Bisha and electron micro- glutaraldehyde in 0.1 M phosphate scopy showed the GO to be present buffer, pH 7.4. Midrib pieces from in these trees. about five to 10 leaves were placed Mexican lime proved to be less in the same tube for each tree. susceptible to greening than sweet The peduncular end of the fruit orange and mandarin. In the affect- axis is rich in phloem tissue and is ed areas, lime trees show greening the best material for GO detection. symptoms (leaf mottle), and elec- The columella tissue was dissected tron microscopy indicates the pres- from the fruit with a razor blade, ence of the GO, yet the trees reach chopped into 2-4 mm long frag- a relatively good size. An addi- ments and fixed. tional reason for fairly good The samples remained in 2% growth is that tristeza disease is glutaraldehyde for 2 to 12 days not present. This is a remarkable until they reached the Bordeaux situation since in Africa and Asia, laboratory where they were post- greening is always accompanied by fixed in 1% osmium tetroxide in tristeza. the above buffer. After dehydra- In the above areas, high per- tion in ethanol, specimens were em- centages of trees are affected. This bedded in Epon and thin sectioned. is correlated with high populations Ultra-thin sections were examined of the Asian vector of greening, Stubborn, Greening and Related Diseases

Fig. 1. Arabian peninsula with some of the main towns. International boundaries are approximate or in some cases still to be settled. Names of towns where greening occurs are underlined. D. citri. The presence of this psylla D. citri, and the disease was was first observed in 1972 and re- probably introduced with D. ported in 1974 (12) on citrus in citri from an Asian country the Djeddah, Wadi Kaulais, Mecca possibly by pilgrims ; and Hadat Ash Shan areas. How- 2) The daily summer temperatures ever, even though it has been known are well above 32°C even at the since 1966 that D. citri is a vector elevation of Taif and Asian of greening (5, 6, 9), no thought greening could survive in this was given until 1981 in Saudi hot climate ; and Arabia to the possibility that the 3) The destruction of sweet orange greening might also be present in and mandarin trees suggests areas where the vector occurred. that the disease must be the se- In certain areas (Taif, Tura- vere, Asian form. bah) the psyllas have become a We conclude that citrus other real pest and cause dieback and se- than Mexican lime has disappeared vere leaf dwarfing and curling. In- or is dying as a result of greening secticide treatments have been in the areas mentioned above. A totally ineffective. high percentage of the Mexican The form of greening present lime trees are affected, but because in Saudi Arabia is very probably of their relative tolerance to green- the Asian form for the following ing and the absence of tristeza, reasons : these trees achieve a certain de- 1) The vector is the Asian psylla, velopment. These numerous rela- 112 Ninth ZOCV Conference

Fig. 2. Electron micrographs of greening organisms (GO) in citrus from Saudi Arabia (A, B and C) and North Yemen (D). A and B) Sieve tubes of sour orange columella (Taif). Upper sieve tube is packed with GO. (A = X 5,000; B = X 20,000). C) GO in sieve tube of Mexican lime leaf (Turabah) (X 5,000). D) GO in sieve tube of sweet orange leaf (Tiaz) (X 55,000). The characteristic ultrastructure of the GO envelope is clearly apparent in C and D. Stubborn, Greening and Related Diseases tively well developed and GO- sweet orange are badly affected, affected lime trees are vast reser- but not killed. voirs of GO inoculum and excellent Greening, a potential danger hosts for the Asian psylla, D. citri. for citrus in the Arabian Peninsu- Ideal conditions for transmission la and the Mediterranean countries. and spread of greening thus exist The presence of Asian greening (a large reservoir of inoculum and and D. citri in Saudi Arabia within a large population of vectors). the oases of the triangle Djeddah- African form of greening dis- Khurmah-Bisha and perhaps be- ease and T. erytreae in North yond this triangle, and of African Yemen. Advanced symptoms of greening and T. erytreae in the ele- greening disease were observed in vated citrus areas of Yemen, is April 1982 on many trees of the not only detrimental to citrus in citrus collection at the Agricultural these areas, but it is also a high Research Station in Taif (Aussfera potential danger to other citrus farm). The trees of this collection areas of the Arabian Peninsula, as were planted in 1974. Leaf and well as to the large citrus growing fruit samples taken from badly regions around the Mediterranean affected sweet orange trees were sea. examined by electron microscopy at The Najran oasis (fig. 1) of the Bordeaux laboratory, and all Saudi Arabia, immediately North were found to contain greening of Yemen, seems well suited for organisms (GO) (fig. 2 D). These citriculture, and projects for citrus observations indicate that the dis- development are underway. The ease affecting these trees is green- area was still free of psyllas and ing. The trees tested negatively for greening as of November 1981, but tristeza virus by ELISA. Symp- both the disease and its Asian toms of greening (yellow branch) vector are present at Bisha only were also observed at Haman Ali 300 km north of Najran. It is even in the Anis region. possible that the disease is closer The areas where greening was than that, since the area between seen were all located at an eleva- Bisha and Najran has not yet been tion of 1000 m and more. No signs surveyed. of the disease were found in the The Unayzah-Buraydah (fig. 1) coastal Tihama areas examined area is also free of greening and (Garouba, Wadi Sardut, Jaraba) . psylla vectors. Uncontrolled move- The geographical distribution of ment of citrus could introduce both the disease reflects the distribution the citrus psyllas and the disease. of the insect vector and in North The coastal Tihama areas south Yemen the vector is the heat sensi- of A1 Hudaydah (the northern tive African psylla, T. erytreae. areas were not surveyed) in Yemen This psylla is known to exist in are free of greening and psylla neighbouring Ethopia and has vectors. Several important citrus very probably been introduced projects are being developed at with citrus from there into the Garouba, Wadi Sardut, and Jaraba. southwestern part of the Arabian The Asian psylla if introduced from Peninsula. Saudi Arabia could entirely ruin The form of greening present the efforts that are now being de- is very probably the heat sensitive voted to citrus in the Tihama. African form since the insect vector The D jeddah-Mecca-Taif area is is the African psylla, the disease only 1000 km south of the eastern and the psylla vector only occur in Mediterranean area and Egypt is cool, elevated areas, and suscept- just across the Red Sea. The ible species and varieties such as Mediterranean basin is free of 114 Ninth ZOCV Conference greening and citrus psyllas and it vectors. Insecticide treatments is very probable that T. erytreae have proven inadequate. It is there- and especially D. citri would be fore proposed that integrated able to multiply if introduced in control of the citrus psyllas be the Mediterranean regions. It is initiated in Saudi Arabia and Ye- highly likely that the disease would men along lines successfully con- be introduced along with the vector. ducted in Reunion Island (1, 2, 7, The introduction of greening and 8). D. citri would be extremely detri- We have also found recently mental to the Mediterranean citrus (December 1983) greening in the industry and must be avoided by all southwest part of Saudi Arabia means. (Abha, Khamis Mushayt) . The The spread of greening within two psyllas were present in this the Arabian Peninsula and towards area indicating that Tr-ioxa ery- the Mediterranean area can only be treae has moved across the border controlled by controlling the psylla up north into Saudi Arabia. LITERATURE CITED 1. AUBERT, B., J. M. BOVE, and J. ETIENNE 1980. La lutte contre la maladie du "greening" des agrumes B I'ile de la RBunion. RBsultats et perspectives. Fruits 35 (10) : 605-624. 2. AUBERT, B., and S. QUILICI 1983. Biological control of the african and asian citrus psyllids (Hemiptera: psylloidae), with eulophid and encyrtid parasites (Hymenoptera: chalci- doidea) in Reunion Island., u. 100-108. In Proc. 9th Conf. IOCV. IOCV, ~iverside. 3. BOVE, J. M., E. C. CALAVAN, S. P. CAPOOR, R. E. CORTEZ, and R. E. SCHWARZ 1974. Influence of temperature on symptoms of California stubborn, South Africa greening, India citrus decline, and Philippines leaf mottling diseases, p. 12-15. In Proc. 6th Conf. IOCV. IOCV, Riverside. BOVE, J. M., P. BONNET, M. GARNIER, and B. AUBERT 1980. Penicillin and tetracycline treatments of greening disease-affected citrus plants in the glasshouse, and the bacterial nature of the procaryote as- sociated with greening, p. 91-102. In Proc. 8th Conf. IOCV. IOCV, River- side. CAPOOR, S. P., D. B. RAO, and S. M. VISWANATH 1967. Diaphorina citri Kuway, a vector of the greening disease of citrus in India. Indian J. Agr. Sci. 37: 572-576. CELINO, C. S., A. A. SALIBE, and R. E. CORTEZ 1966. Diaphorina citri Kuway, the insect vector for the leaf mottle virus of citrus in the Philippines. Bureau of Plant Industry, Manila, Philippines. Sci. Rev. Philippines 10 (4) : 27-29. ETIENNE, J. 1978. Contrhle biologique 2 la RBunion de Trioxa erytreae (Homopt. Psyllidae) au moyen de Tetrastichus drgi (Hym. Eulophidae). Fruits 33(12) : 877- 882. ETIENNE, J., and B. AUBERT 1980. Biological control of psyllid vectors of greening disease in Reunion Island, p. 118-121. In Proc. 8th Conf. IOCV. IOCV, Riverside. MARTINEZ, A. L., and J. M. WALLACE 1967. Citrus leaf-mottle yellows in the Philippines and transmission of the causal virus by a psyllid Diaphorina citri. Plant Dis. Rep. 51: 692-695. MASSONIE, G., M. GARNIER, and J. M. BOVE 1976. Transmission of Indian citrus decline by Trioxa erytreae (Del G.), the vector of South African greening, p. 18-20. In Proc. 7th Conf. IOCV. IOCV, Riverside. McCLEAN, A. P. D., and P. C. J. OBERHOLZER 1965. Citrus psylla, a vector of the greening disease of sweet orange. S. Afr. J. Agr. Sci. 8: 297-298. WOOLER, A., D. PADGAM, and A. ARAFAT 1974. Saudi Arabia Diaphorina citri on citrus. FA0 Plant Protection Bulletin 22 (4) : 93-94.