World citrus production in 1995/1996 was expected to increase, by around 3 %, to a record of nearly 80 million tones, including all varieties. The expected increase, nearly 18 %, was mainly due to a larger orange crop in Brazil, that reflected abundant supplies after a weather-reduced harvest of 94/95. São Paulo and Florida are the two mainly citrus growing areas of the world. In an area of approximately 630.200 ha São Paulo has 164 million trees, and an annual production of 374 millions of orange boxes (40,8 kg), corresponding to 87 and 30 % of the Brazilian, and of the world production, respectively. Over 70 % of orange produced in São Paulo supply the orange concentrate juice companies. With increasing participation the internal markets consume around 28 % of the fresh fruit production. Although being the world bigger producer, Brazil has small presence in the fresh fruits exportation market.

São Paulo and Florida also account for 90 % of world concentrate (61º Brix) orange juice production (2.138,5 thousand tons), 1.146,9 and 872 thousand tons, respectively. There are 22 orange juice industries in São Paulo with a total of 994 reamer-type extractors. The citrus industries in São Paulo employ around 400,000 people distributed in 204 cities, and 20,000 growers. The more important importers of concentrate orange juice from Brazil in 1995/1996 were European Union (68,8 %), North America (18,5 %), Asia (9,5 %) and other countries (3,2 %).

The citriculture in São Paulo stays unique in the analysis of the development of the Brazilian agriculture in the last 30 years. From supplier of the internal Brazilian market, and with incursions in fresh fruit exports until the beginning of the 60’s, the citrus industry in Brazil developed itself, becoming in the beginning of the 80’s the main citrus producer, and the biggest exporter of frozen concentrate, reaching annual exchange values on the order of 1,2 to 1,4 billions of dollars.

Although the soil and clime condition in several areas of Brazil enable growing citrus, our productivity still remain very low comparing with other countries (2.0 boxes/tree/year in Brazil, and 6,0 boxes/tree/year in Florida). The increase of production in last years is correlated to spreading of new areas. The main factors associated with the low productivity are diseases, pests, water deficit, and nutritional factors.

The CVC was first related in 1987 in São Paulo and Minas Gerais States affecting all commercial sweet orange varieties. Following the initial observations, the disease spread rapidly by graft propagation with infected budwood and by sharpshooter vectors, and became widely distributed in all citrus growing regions of Brazil. CVC is now a major concern to the Brazilian citrus industry and is considered to be potentially the more devasting citrus disease. CVC has been found only in Brazil and Argentina.

Disease symptoms include mottled and interveinal chlorosis similar to that seen associated with zinc deficiency, reduced leaf size, and fruit that are small, early maturing, and have a very hard rind which can damage juicing machines (Fig. 1). Young leaves do not show symptoms. As the leaves muture, small light brown gummy lesions appear on the under side of the leaves which correspond to the yellow chlorotic areas on the leaf upper side. The lesions on the under side of the leaves may become dark brown or even necrotic (Fig. 2).

Fig. 1: Severe symptoms of CVC on an three years old tree of sweet orange ‘Pera’.

Fig. 2: Typical leaf symptoms of CVC in sweet orange.

In newly affected trees, symptoms may affect only a tree sector, whereas trees which have been infected for a period of time show variegated chlorosis throughout the canopy. After infection with the bacteria the symptoms can take more than one year to be observed. The earlier young plants are infected, the more severe are the symptoms. Infected plants in the nursery or early in the field do not survival two years. All sweet orange varieties are susceptible; mandarins, lemons, and some commercial hybrids are considered to be tolerant.

The last surveys have shown the progress of CVC through São Paulo State. The North, Northwest and Center regions have higher incidence. The surveys in 1992 showed 64 % of groves with at least one tree with visual symptoms. In 1996, in 88 % of the orchads were found plants (3 to 5 years old) with severe symptoms of CVC. Epidemiological studies estimate that CVC from a single infected tree can spread to 90 % of the trees in a 20 hectare grove in 12 years.

Xylem feeding homopterans, such as sharpshooter leafhoppers (Homopetera, Cicadellidae, subfamily Cicadellinae) are vectors of X. fastidiosa. The bacterium multiplies and attaches to the food canal (precibarium) and pumping chamber (cibarium) in the foregut of the vector insect. Large number of bacteria can accumulate in the foregut over time. In Brazil the bacteria of CVC were found associated with Oncometopia facialis, Acrogonia terminalis, and Dilobopterus costalimai. Other species of sharpshooters can also be involved in the transmission. Infected propagation material (budwoods) and infected plants in the nursery are the main way for spreading the disease at long distances.

At short time the current possibilities of control include pruning of infected branches, chemical control of the vectors, and/or rouging of severe affected plants. For new orchads the use of bacteria-free budwoods and healthy plants seem to be the only way to insure the production. Selection of tolerant or resistant varieties using massal selection in high effected groves or in germplasm collections, and sexual hybridization for new genetic combinations are approaches that have been used for a long term solution of CVC.

The term ‘xylem-limited bacteria’ (XLB) was used to describe prokaryotic plant pathogens difficult to isolate by standard bacteriology procedures. These fastidious organisms require complex media for growth, occur only in the xylem of infected plants, are transmitted by xylem-feeding leafhoppers, and cause difficult-to-control diseases of economically important crops. Due to ultrastructural similarities to animal rickettsiae they were first referred to as ‘rickettsia-like bacteria’. XLB have been found to be unrelated to this group or to other plant pathogens. XLB are rod-shaped with distinctive rippled cell walls, nonflagellate, do no form spores, measure 0,3 - 0,5 m m in diameter, and 1-5 m m in length. The fastidious bacteria grow well at 20 - 25 oC and pH 6,7 - 7,0. Studies with various XLB show the G + C ratio to be about 50,5 mol%, with genome molecular weights of 1,4 + 0,2, or 1,92 - 2,42 Mb. No genetic relatedness has been shown between XLB and other plant-pathogenic bacteria. Although all fastidious, Gram-negative, xylem-limited bacteria were included in the species Xyella fastidiosa, there is enough strain variability to justify taxonomic separation at the subspecies or pathovar level.

Xylella fastidiosa is the causal agent of many economically important plant diseases including Pierce’s disease of grapevine (PD), alfalfa dwarf, phony peach disease (PPD), periwinkle wilt (PW), leaf scorch of plum, mulberry, pear, almond, elm, sycamore, oak, maple, coffee, and citrus variegated chlorosis (CVC). Until 1973 many that diseases were attributed to a virus, strengthened by detection of insect vectors. Research was hindered by the fact that X. fastidiosa is often present in asymptomatic plants and was difficult to isolate and grow in axenic culture. The bacterium of PD was the first to be cultured in 1978. Pathogenic relationships among strains of X. fastidiosa are not very well characterized and seem to be quite complex. DNA analysis suggests the existence of five groups of X. fastidiosa: the citrus group, the plum-elm group, the grape-ragweed group, the almond group, and mulberry group.

There is considerable disagreement on the mechanism of pathogenesis in diseases caused by X. fastidiosa. Dysfunction of the water-conducing system, phytotoxin production, and growth regulator imbalance are the proposed mechanisms of pathogenesis. Evidences support the hypothesis that the mechanism of pathogenesis is the production of water stress due to vascular occlusions (bacterial aggregates, host gum, and tyloses). X. fastidiosa must have special mechanisms to concentrate and absorb nutrients from a the environment. Aggregate of bacteria appeared to be attached to vessel walls by extracellular strands produced by the bacteria that are usually most abundant at the end of the bacterial rods. The strands resemble the polysaccharide fibers that constitute the "glycocalyx", which is thought to attach bacteria in a variety of turbulent habitats such as rapidly flowing streams or animal guts. The negatively charged polysaccharide fibers of the glycocalyx may function as an ion-exchange substrate, binding nutrient ions to the bacterial aggregate or conserving and concentrating digestive enzymes released by the bacteria for action against the host tissue.

The basic and important question of how X. fastidiosa spread within the xylem system is unanswered. The use of reporter genes to aid in detecting the location of bacteria within living plants would facilitate in vivo studies of their movements within plants. Micrographs of vascular tissues from various plant organs colonizeid by X. fastidiosa show that movement of the bacterium from cell to cell is restrained by xylem pit membranes. The movement of bacteria from vessel to vessel suggests that they produce enzymes that degrade the pit membrane. Symptoms in plants appear to depend on the rate and extent of colonization. The relationship of bacterial population and bacterial toxins are probably not X. fastidiosa.

The most used diagnostic procedures for X. fastidiosa include serological methods (ELISA/DIBA, and immunofluorescence) that detect around 10⁴ bacteria/ml, and DNA based methods (PCR, RFLP, and analysis of plasmids) which sensibility can reach 10² bacteria/ml.

Most of the economically important hosts of X. fastidiosa are long-lived perennials requiring long periods of time for fruit production and conventional breeding for resistance. Traditional cultivars (grapes, citrus, etc) are crucial to marketing the crop. This makes genetic engineering for improving resistance of popular cultivars a speculative but promising altenative to conventional breeding.

Each plant pathogen has a unique mode of patogenicity. Despite the vast array of potential phytopathogens, resistance (lack of susceptibility) is the rule and susceptibility is the exception. Resistance to pathogen is manifested in a variety of ways and is often correlated with a hypersensitive response (HR), localized induced cell death in the host plant at the site of infection. Resistance does not always involve visible HR, which may reflect either HR limited to individual plant cells or other uncharacterized defense mechanisms. Alternatively, the pathogen could lack a specific pathogenicity function required to cause disease in the host, or the host could lack a specific ‘susceptibility’ factor.

The theoretical basis for the gene-for-gene hypothesis of plant-pathogen interaction and for molecular cloning for pathogen avirulence genes and their corresponding plant R genes was first provided by H.H. Flor (1971). An avr (avirulence) gene gives the pathogen an avirulent phenotype on a host plant that carries the corresponding R (resistance) gene. The receptor-ligand model postulates that pathogen avr gene specify elicitor molecules that induce disease resistance in host plants that contain a cognate R gene. Some avirulence gene avr has been identified in bacteria. Why do pathogens contain avirulence genes ? The avr gene may encodes pathogenicity factors that confer a selective advantage for the pathogen, as has been shown for several bacterial avirulence genes that confer enhanced virulence on susceptible hosts, that is, on hosts that do not carry a cognate R gene.

The cloning and characterization of several plant R (resistance) genes constitutes a major breakthrough in the elucidation of the molecular basis of disease resistance to a wide range of phytopathogens. As a result, we are finally in a position to determine the molecular basis of plant pathogen specificity and expression of disease resistance. Future research challenges include the determination of the mechanisms by which R gene products recognize pathogen elicitors and the plant defense response blocks pathogen growth. The basic knowledge obtained from this research will undoubtedly help to produce novel forms of durable disease resistance and will lead to a decline in the use of environmentally damaging pesticides.

The main questions for using such approaches in the disease citrus variegated chlorosis (CVC) is the absolute lack of information about the system X. fastidiosa in citrus. Several plant-pathogen interaction models have been improved, and could be used too. Questions like genetic diversity among strains, the real size of genome, the occurrence of plasmids, and their association with pathogenicity, the occurrence of avirulence or virulence genes that could be associated with the development of symptoms, or with the vectors, are questions that a genome project will help to answer.

• Beretta, M.J.G., R.F. Lee, K.S. Derrick, C.L. Davis & G.A. Barthe. 1992. Culture and serology of a Xylella fastidiosa associated with citrus variegated chlorosis in Brazil. Proc. Int. Soc. Citriculture 2: 830-831.

- Maria Julia Gobbo Beretta, Seção de Bioquímica Fitopatologica, Instituto Biológico de São Paulo, Tel. 011 - 572 - 9822 / 549 0114

- R.F. Lee, University of Florida, IFAS, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred 33850 / USA. Tel. 941 - 956 1151. Fax. 941 - 956 4631. E-mail: rfl@icon.lal.ufl.edu

- K.S. Derrick, University of Florida, IFAS, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred 33850 / USA. Tel. 941 - 956 1151. Fax. 941 - 956 4631. E-mail: ksd@icon.lal.ufl.edu

• Hartung, J.S. M.J. G. Beretta, R.H. Brlansky, J. Spisso & R.F. Lee. 1994. Citrus variegated chlorosis bacterium: axenic culture, pathogenicity, and serological relationship with other strains of Xylella fastidiosa. Phytopatology 84: 591-597.

- Dr. J.S. Hartung, Fruit Laboratory, Agricultural Research Service, USDA, Beltsville Agriculture Research Center, Building 010A, Beltsville, MD 20705, USA. Tel. 301 - 504-6374, Fax. 301 - 504-5062. E-mail: jhartung@asrr.arsusda.gov

- M.J.G. Beretta (see above)

- R.H. Brlansky, University of Florida, IFAS, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred 33850 / USA.

- R.F. Lee (see above)

• Hill, B.L. & A.H. Purcell. 1995. Acquisition and retention of Xyella fastidiosa by an efficient vector, Graphocephala atropunctata. Phytopatology 85: 209-212.

- A.H. Purcell, Department of Environmental Science, Policy and Management, University of California, Berkeley 94720-3112. E-mail: purcell@nature.berkely.edu

• Hopkins, D.L. Xylella fastidiosa: xylem-limited bacterial pathogen of plants. 1989. Ann. Rev. Phytopatol. 27:271-290.

- D.L. Hopkins, University of Florida, Central Florida Research and Education Center, Leesburg 32749-0388.

• Leite Jr., R.P., D.S. Egel & R.E. Stall. 1994. Genetic analysis of hrp-related DNA sequences of Xanthomonas campestris strains causing diseases in citrus. Applied and Environ. Microbiology 60: 1078-1086.

• Machado, M.A., M.L.P.N.Targon, M.J.G. Beretta, F.F. Laranjeira & S. A. de Carvalho. Detecção de Xylella fastidiosa em diferentes espécies e variedades de citros sobre-enxertadas em laranja Pera com clorose variegada dos citros (CVC). Fitopatologia Brasileira

- M.A.Machado, Centro de Citricultura Sylvio Moreira, C.P. 04 - CEP 13490-970 Cordeirópolis/SP - Tel/Fax. 019 -5461399. E-mail: fiac@siteplanet.com.br

• Pooler, M.R. & J. S. Hartung. 1995. Genetic relationship among strains of Xylella fastidiosa from RAPD-PCR data. Current Microbiology 31: 134-137.

• Pooler, M.R. & J. S. Hartung. 1995. Specific PCR detection and identification of Xylella fastidiosa strains causing citrus variegated chlorosis. Current Microbiology 31: 377-381.

- J.S. Hartung (see above)

• Purcell, A.H. & D.L. Hopkins. 1996. Fastidious xylem-limited bacterial plant pathogens. Annu. Rev. Phytopathol. 34: 131-151.

• Rajua, B.C. & J.M. Well. 1986. Diseases caused by fastidious xylem-limited. Plant Disease 70: 182-186.

- B.C. Raju, Weyerhaeuser Tissue Culture Center, Apopka, Florida

- J.M. Wells, USDA, Horticultural Crops Quality Research Laboratory, Rutgers University, New Brunswick, New Jersey / USA.

• Rossetti, V. M. Garnier, J. M. Bové, M.J.G. Beretta, A.R.R. Teixeira, J.A. Quaggio & J. Dagoberto de Negri. 1990. Présence de bactéries dans le xylème d’oranges atteints de chlorose variégée, une nouvelle maladie des agrumes au Brésil. C.R. Acad. Sci., Paris, 310: 345-349.

- V. Rossetti, Instituto Biológico de São Paulo,

- M. Garnier, Laboratories de Biologie Cellularaire & Moléculaire, INRA & Universit’t de Bordeaux III, B.P. 81, 33883 Villenave d’Ornon Cedex, France.

- J.M. Bové (see M.Garnier)

- J.A. Guaggio, Seção de Fertilidade do Solo, Instituto Agronômico de Campinas.

- D. de Negri, Coordenadoria de Assistência Técnica Integral (CATI), Campinas.