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Data sheets on quarantine pestsPrepared by CABI and EPPO for the EU under Contract 90/399003 Citrus greening bacterium
Name: Citrus greening bacterium
Taxonomic position: Bacteria: Gracilicutes
Common names: Greening (Africa), leaf mottling (Philippines), decline (India), vein
phloem degeneration (Indonesia) (English) Huang long bin (yellow shoot), likubin (decline) (Chinese) Greening (French) Enverdecimiento (Spanish) Notes on taxonomy and nomenclature: A fastidious, phloem-limited Gram-negative
bacterium, which has not been cultured. Two forms occur: a heat-tolerant (Asian) form and
a heat-sensitive (African) form. Jagoueix et al. (1994) have recently proposed the genus
Liberobacter for the greening pathogen, considered to be a member of the alpha
subdivision of the Proteobacteria on the basis of PCR studies. The names L. asiaticum and
L. africanum have been proposed for the two forms, which can be distinguished as separate
species on the basis of sequence homology (Planet et al., 1995). However, the names have
yet to be accepted as valid.
Bayer computer code: CSGXXX
EPPO A1 list: No. 151
EU Annex designation: II/A1
Citrus greening bacterium infects citrus generally. The bacterium may persist and multiply in most Citrus spp. but most severe symptoms are found on oranges (C. sinensis), mandarins (C. reticulata) and tangelos (C. reticulata x C. paradisi). Somewhat less severe symptoms are found on lemons (C. limon), grapefruits (C. paradisi), C. limonia, C. limettioides, rough lemons (C. jambhiri), kumquats (Fortunella spp.) and citrons (C. medica) (McClean & Schwarz, 1970). Symptoms are even weaker on limes (C. aurantiifolia) and pummelos (C. grandis). Though other Rutaceae have been infected artificially, there are not apparently any records of their natural infection. Citrus greening bacterium has been experimentally transmitted, by Cuscuta campestris, from citrus to one non-rutaceous host Catharanthus roseus (Garnier & Bové, 1983). GEOGRAPHICAL DISTRIBUTION
The distribution is given separately for the two forms of the disease. • Heat-sensitive form ("Liberobacter africanum")
EPPO region: Absent.
Asia: Saudi Arabia, Yemen.
Africa: Burundi, Cameroon, Central African Republic, Comoros, Ethiopia, Kenya,
Madagascar, Malawi, Mauritius, Réunion, Rwanda, Somalia, South Africa, St. Helena
(unconfirmed), Swaziland, Tanzania, Zimbabwe. Recent records are from Aubert et al.
(1988). Surveys have failed to find the disease in Gabon, Namibia and Zambia.
• Heat-tolerant form ("Liberobacter asiaticum")
EPPO region: Absent.
Asia: Bangladesh, China (Guangdong, Guangxi), Hong Kong, India (Arunachal Pradesh,
Assam, Haryana, Maharashtra, Manipur, Meghalaya, Punjab, Sikkim, West Bengal),
Indonesia (Java, Sumatra), Japan (Ryukyu Islands), Malaysia (Peninsular), Nepal, Pakistan,
Philippines, Saudi Arabia, Taiwan, Thailand, Viet Nam.
Africa: Mauritius, Réunion.
Note that both forms exist in Mauritius, Réunion and Saudi Arabia. The Mediterranean area
and most of the Middle East (e.g. Iran) are still free from the disease. The infested area
which is closest to the Mediterranean zone extends south of Mecca, along the Red Sea.
Distribution map: See Commonwealth Department of Health (1982).
Graca (1991) has recently provided a comprehensive review of the disease. Transmission of the greening agent by graft inoculation was first reported in China (Lin, 1956). The agent was initially thought to be a phytoplasma, but later work showed it to be a bacterium with a peptidoglycan-containing membranous cell wall of the Gram-negative type (Moll & Martin, 1973; Garnier et al., 1984). The bacterial nature of the greening organism explains why penicillin treatment of infected plants results in symptom remission (Aubert & Bové, 1980; Bové et al., 1980). A report claiming culture of citrus greening bacterium has appeared (Garnett, 1985). There is, however, as yet, no experimental evidence to show that the cultured organism is really the greening bacterium. Two forms of greening disease are known (Bové et al., 1974). One, in the southern part of Africa, is heat-sensitive, as symptoms do not develop in hot climates where temperatures above 30°C are reached several hours a day. The other form is heat-tolerant and withstands high temperatures, and is predominantly Asian in distribution. The Asian heat-tolerant form of the disease has been discovered in Saudi Arabia, and the African heat-sensitive form in Yemen. When the African and the Asian forms of the greening bacterium were transmitted from citrus to Catharanthus roseus by Cuscuta campestris (Garnier & Bové, 1983), the African form remained heat-sensitive and the Asian form heat-tolerant in both C. roseus and citrus. Monoclonal antibodies to citrus greening bacterium and more specifically to an isolate from India, have been obtained (Garnier et al., 1987). Using immunofluorescence on sections, the monoclonal antibodies reacted not only with the homologous (Indian) isolate of citrus greening bacterium, but also with isolates from the Philippines, Réunion and elsewhere in Africa. There are therefore serological relationships between African and Asian forms of citrus greening bacterium, but it is possible that further work will discover strain differences. Under natural conditions greening is transmitted in Africa and Yemen by Trioza erytreae (McClean & Oberholzer, 1965; EPPO/CABI, 1996b) and in Asia (including Saudi Arabia) by Diaphorina citri (Capoor et al., 1967; EPPO/CABI, 1996a). However, it was shown experimentally that T. erytreae can transmit the Indian form of greening (Massonie et al., 1976) and that D. citri is able to transmit the African form (Lallemand et al., 1986). DETECTION AND IDENTIFICATION
The general aspect of affected trees is open growth, stunting, twig dieback, sparse yellow
foliage, severe fruit drop; on certain trees and in certain countries (China), symptoms are
seen initially on one limb of an affected tree (yellow branch aspect); severe decline is
mainly seen with Asian greening.
Some fruits are under-developed, lopsided, and poorly coloured. When pressure is exerted
with a finger, a greyish-white waxy mark appears sometimes on the rind surface. Seeds are
often aborted. The greening symptom, mainly occurring in Africa, is seen on fruits which
mature only on the side exposed to the sun, the unexposed side remaining dull olive-green
(Commonwealth Department of Health, l982).
Mottling and zinc deficiency-like symptoms are the most common and characteristic.
Mature leaves often show irregular patches between the main veins. The veins are often
prominent and yellow.
On trunk, limbs and shoots
No symptoms apparent.
Localized zones of necrotic phloem are scattered through the vascular system of the leaf.
Massive accumulation of starch in the plastids is seen together with aberrations in cambial
activity and excessive phloem formation.
Elongated sinuous rod-like structures, 0.15-0.25 µm in diameter and µm long. These can be
seen by electron microscopy in the sieve tubes of infected trees. Similar structures have
been seen in both vectors.
Detection and inspection methods
Suspect material may be grafted onto sensitive indicator plants. Preferred indicator plants
are Orlando tangelo and sweet orange seedlings. Inoculation should preferably be with
pieces of mottled leaves. Because of the variable results in graft transmission, at least ten
seedlings should be used for each tree to be indexed. After inoculation, the indicator
seedling should be kept at 24°C (South African form) or 32°C (Asian form). Symptoms
usually show after 4-5 months. The presence of a specific fluorescent marker, gentisoyl
glucoside (Feldman & Hanks, 1969), in greening-infected tissue has been used for
detection (Schwarz, 1968a; 1968b). The reliability of this method was further confirmed by
Hooker et al. (1993). Suspect trees may be analysed by electron microscopy to confirm the
presence of the characteristic bacteria in the sieve tubes. Serological identification using
monoclonal antibodies against the Indian form of citrus greening bacterium has been
successfully used to detect the bacterium in greenhouse-grown citrus and Catharanthus
roseus by immunofluorescence and ELISA (Garnier et al., 1987). DNA probes have
recently been obtained which detect the pathogen and differentiate between its two forms
(Villechanoux et al., 1992).
MEANS OF MOVEMENT AND DISPERSAL
Citrus greening bacterium can be moved by its vectors or in citrus plants for planting. Seed transmission does not occur. Since the two known psyllid vectors of the disease are not present in the Mediterranean area, parts of the Middle East and America, the disease, if introduced into these areas, should in theory only spread by propagation of infected plant material. However, it is conceivable that hitherto unrecognized vectors of greening exist. PEST SIGNIFICANCE
Citrus greening, transmitted by D. citri and T. erytreae, is an extremely severe disease. In
South Africa, in 1965, fruit losses from the disease were 30-100% in individual orchards;
many of these had subsequently to be abandoned or removed. Earlier outbreaks occurred in
l932-l936 and 1939-1946. Annual losses in 1991 are estimated at ZAR 35 million. In
Réunion, large areas of citrus cultivation had to be abandoned (Catling, 1973), and also in
Thailand (Schwarz & Knorr, 1973). In the Philippines, mandarin production fell from
11700 t in 1960 to 100 t in 1968. In south-western Saudi Arabia, sweet orange and
mandarin practically disappeared during the 1970s. In Asia, an FAO-UNDP programme
has recently been established to try to control the disease.
In the Transvaal (South Africa), citrus is treated against greening by injection of
tetracycline (up to 20 g per adult tree) with high-capacity compressors working at 10
kg/cm2. Such methods have been tried, but not widely used, in Asia. Treatment with
rolitetracycline also reduces symptom expression. However, the main emphasis of control
in South Africa is based on the use of healthy nursery trees and effective systemic
insecticides (against the vector T. erytreae) as trunk treatments (Buitendag, 1991). Santokh
Singh et al. (1994) have obtained promising results using cross protection with mild strains
of greening, but this approach has not been used in practice.
Citrus greening bacterium is listed as an A1 quarantine pest by EPPO (OEPP/EPPO, 1988)
and is also of quarantine significance for COSAVE, CPPC and IAPSC. In the citrus-
growing areas of the EPPO region, it could only present a problem if introduced with one
or other of its vectors. There is no suggestion that native Mediterranean vectors could exist.
In view of its severity it is essential to keep the disease and its vectors out of the
Mediterranean zone and to prevent their spread in the Middle East. D. citri and T. erytreae
are also considered as A1 quarantine pests by EPPO (OEPP/EPPO, 1988; EPPO/CABI,
1996a, b), in their own right as well as because of their vector status.
EPPO recommends (OEPP/EPPO, 1990) that importation of plants for planting and cut branches of citrus from countries where citrus greening bacterium or either of its vectors occur should be prohibited. Healthy budwood can be obtained from material under quarantine by shoot-tip grafting, or alternatively by heat treatment (e.g. water-saturated hot air at 49°C for 50 min) of budwood in tetracycline solution (1000 p.p.m.), which results in a high percentage of greening-free buds. Various other heat-treatment regimes are also possible. Such greening-free material should be kept and propagated under insect-proof screenhouse conditions, and its health status checked by grafting onto sweet orange. BIBLIOGRAPHY
Aubert, B.; Bové, J.M. (1980) Effect of penicillin or tetracycline injections of citrus trees affected by greening disease under field conditions in Réunion Island. In: Proceedings of the 8th Conference of the International Organization of Citrus Virologists (Ed. by Calavan, E.C.; Garnsey, S.M.; Timmer, L.W.), pp. 103-108. University of California, Riverside, USA. Aubert, B.; Garnier, M.; Cassim, J.C.; Bertin, Y. (1988) Citrus greening disease in east and west African countries south of the Sahara. In: Proceedings of the 10th Conference of the International Organization of Citrus Virologists, pp. 226-230. Bové, J.M.; Calavan, E.D.; Capoor, S.P.; Cortez, R.E.; Schwarz, R.E. (1974) Influence of temperature on symptom of Californian stubborn, South African greening, Indian citrus decline and Philippines leaf mottling disease. In: Proceedings of the 6th Conference of the International Organization of Citrus Virologists (Ed. by Weathers, L.G.; Cohen, M.), pp. 12-15. University of California, Berkeley, USA. Bové, J.M.; Bonnet, P.; Garnier, M.; Aubert, B. (1980) Penicillin and tetracycline treatments of greening disease affected citrus plants in the glasshouse and the bacterial nature of the prokaryote associated with greening. In: Proceedings of the 8th Conference of the International Organization of Citrus Virologists (Ed. by Calavan, E.C.; Garnsey, S.M.; Timmer, L.W.), pp. 91-97. University of California, Riverside, USA. Buitendag, C.H. (1991) The current status and the control of greening disease of citrus in the Republic of South Africa. Citrus Journal 1, 35-40.
Capoor, S.P.; Rao, D.B.; Viswanath, S.M. (1967) Diaphorina citri, a vector of the greening disease of citrus in India. Indian Journal of Agricultural Science 37, 572-576.
Catling, H.D. (1973) Notes on the biology of the South African citrus psylla Trioza erytreae. Journal of the Entomological Society of South Africa 36, 299-306.
Commonwealth Department of Health (1982) Citrus dieback and greening. Plant Quarantine Leaflet No. 26. Australian Government Publishing Service, Canberra, Australia. EPPO/CABI (1996a) Diaphorina citri. In: Quarantine pests for Europe. 2nd edition (Ed. by Smith, I.M.; McNamara, D.G.; Scott, P.R.; Holderness, M.). CAB INTERNATIONAL, Wallingford, UK. EPPO/CABI (1996b) Trioza erytreae. In: Quarantine pests for Europe. 2nd edition (Ed. by Smith, I.M.; McNamara, D.G.; Scott, P.R.; Holderness, M.). CAB INTERNATIONAL, Wallingford, UK. Feldman, A.W.; Hanks, R.W. (1969) The occurrence of a gentisic glucoside in the bark and albedo of virus-infected citrus trees. Phytopathology 59, 603-606.
Garnett, H.M. (1985) Isolation of the greening organism. Citrus and Subtropical Fruit Journal No. Garnier, M.; Bové, J.M. (1983) Transmission of the organism associated with citrus greening disease from sweet orange to periwinkle by dodder. Phytopathology 73, 1358-1363.
Garnier, M.; Danel, N.; Bové, J.M. (1984) Etiology of citrus greening disease. Annales de Microbiologie (Institut Pasteur) 153A, 169-179.
Garnier, M.; Martin-Gros, G.; Bové, J.M. (1987) Monoclonal antibodies against the bacterial-like organism associated with citrus greening disease. Annales de Microbiologie (Institut Pasteur) 138,
Graca, J.V. da (1991) Citrus greening disease. Annual Review of Phytopathology 29, 109-136.
Hooker, M.E.; Lee, R.F.; Civerolo, E.L.; Wang, S.Y. (1993) Reliability of gentisic acid, a fluorescent
marker, for diagnosis of citrus greening disease. Plant Disease 77, 174-180.
Jagoueix, S.; Bové, J.M.; Garnier, M. (1994) The phloem-limited bacterium of greening disease of citrus is a member of the alpha subdivision of the Proteobacteria. International Journal of
Systematic Bacteriology 44, 379-386.
Lallemand, J.; Fos, A.; Bové, J.M. (1986) Transmission de la bactérie associée à la forme africaine de la maladie du 'greening' par le psylle asiatique Diaphorina citri. Fruits 41, 341-343.
Lin, K.H. (1956) Observations on yellow shoot on citrus. Etiological studies of yellow shoot on citrus. Acta Phytopathologica Sinica 2, 1-42.
Massonie, G.; Garnier, M.; Bové, J.M. (1976) Transmission of Indian citrus decline by Trioza erytreae, the vector of South African greening. In: Proceedings of the 7th Conference of the International Organization of Citrus Virologists (Ed. by Calavan, E.C.), pp. 18-20. University of California, Riverside, USA. McClean, A.P.D.; Oberholzer, P.C.J. (1965) Citrus psylla, a vector of the greening disease of sweet orange. South African Journal of Agricultural Science 8, 297-298.
McClean, A.P.D.; Schwarz, R.E. (1970) Greening or blotchy-mottle disease of citrus. Phytophylactica 2, 177-194.
Moll, J.N.; Martin, M.N. (1973) Electron microscope evidence that citrus psylla (Trioza erytreae) is a vector of citrus greening in South Africa. Phytophylactica 5, 41-44.
OEPP/EPPO (1988) Data sheets on quarantine organisms No. 151, Citrus greening bacterium and its vectors Diaphorina citri & Trioza erytreae. Bulletin OEPP/EPPO Bulletin 18, 497-507.
OEPP/EPPO (1990) Specific quarantine requirements. EPPO Technical Documents No. 1008. Planet, P.; Jagoueix, S.; Bové, J.M.; Garnier, M. (1995) Detection and characterization of the African citrus greening liberobacter by amplification, cloning, and sequencing of the rplKAJL-rpoBC
operon. Current Microbiology 30, 137-141.
Santokh Singh; Kapur, S.P.; Cheema, S.S. (1994) Management of greening disease through cross- protection. Indian Journal of Virology 10, 113-121.
Schwarz, R.E. (1968a) Indexing of greening and exocortis through fluorescent marker substances. In: Proceedings of the 5th Conference of the International Organization of Citrus Virologists (Ed. by Childs, J.F.L.), pp. 118-124. University of Florida Press, Gainesville, USA. Schwarz, R.E. (1968b) Thin layer chromatographical studies on phenolic markers of the greening virus in various citrus species. South African Journal of Agricultural Science 11, 797-801.
Schwarz, R.E.; Knorr, L.C. (1973) Presence of citrus greening and its psylla vector in Thailand. FAO Plant Protection Bulletin 21, 132-138.
Villechanoux, S.; Garnier, M.; Renaudin, J.; Bové, M. (1992) Detection of several strains of the bacterium-like organism of citrus greening disease by DNA probes. Current Microbiology 24, 89-
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