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Fighting the yellow dragon

Studies and solutions to fight greening, the disease that has been attacking Brazilian citriculture since 2003

fundecitrusOrange tree disease makes leaves go yellowish and means plant must be eradicatedfundecitrus

A veritable war is being waged by Brazilian citriculture against greening, currently the most devastating disease in citrus fruit, a vegetable group that comprises orange, lemon, tangerine, lime and grapefruit trees. Identified in 2003 for the first time in this country, it has created a battle field in which, on one side, we have the bacteria that infect that plants, leaving their leaves yellowed and the fruit deformed and unsuitable for consumption. On the opposite side, we have a horde of researchers from a range of Brazilian and international institutions and from Fundecitrus (the Citriculture Defense Fund, an organization maintained by the producers), who are trying to bring to a halt the progress of the disease in the plantations, along with the citrus farmers, in particular in the states of São Paulo and Paraná, and in the South of the state of Minas Gerais. These areas account for almost 90 % of the national production of citrus fruit and 60% of the world production of frozen concentrated juice.

The studies conducted have already enabled the development of molecular tests to identify the sick plants and the establishment of forms of control, such as the eradication of the citrus trees attacked by greening. Furthermore, there are studies under way on how to keep the disease from spreading further. “The infection is severe. It is useless to cut branches off; one has to pull out the tree, including its roots, with a machine, to ensure it won’t sprout up again,” says agronomist Marcos Antônio Machado, a researcher and director of the Sylvio Moreira Citriculture Center, which belongs to the IAC Agronomy Institute and to the Bureau of Agriculture and Supplies, in the town of Cordeirópolis. According to Fundecitrus, more than 4 million trees out of a total of some 200 million in Brazil have already been eliminated, causing immense and varied losses, depending on the age of each plant. An orange tree, for instance, can produce fruit for more than ten years. According to a study conducted in March and April of this year by the Agricultural Defense Coordinating Office, also under the São Paulo Agriculture Bureau, 18% of the São Paulo state plantations have been affected and have at least one tree with greening; this figure has risen by 30% relative to 2008.

Machado was on the research team that identified, in June 2004, in the municipality of Araraquara, in inner-state São Paulo, for the first time in Brazil, the bacteria that causes the disease. The confirmation was conducted using molecular biology techniques, which amplify the bacterial DNA through a polymerase chain reaction (PCR). These tests are now used routinely, both at the Citriculture Center and at Fundecitrus, in order to confirm plant disease. Machado tells us that greening may have come into Brazil through buds, vegetation propagation material, more than ten years ago. “Somebody probably thought a foreign variety was pretty and brought the material into Brazil.” The disease has been reported since the nineteenth century in Asia, the continent where citrus fruit originally came from. Citrus fruit was found especially in India and in China, the latter being the country in which the disease was first described. There, it was named huanglongbing, or HLB, which means “yellow dragon disease.” The term greening was coined in South Africa and became known worldwide. It refers to the fact that the fruit do not ripen and remain green. “We prefer to call it by the official Chinese name, because they were the forerunners of its description,” he says.


Brazilian farmers are highly familiar with the insect that disseminates the bacteria. It got here in the early 1940, though it is not known how, but probably in the midst of infected seedlings. It took to the climate, but was not regarded as a pest because it did not give rise to any losses, although it was connected with transmitting the HLB bacteria in China and in other Asian countries. The attitude of Brazilian citricultures regarding Diaphorina citri, also known to science as a psyllid, which is two to three millimeters long, only changed once greening had been confirmed in São Paulo. It transmits or acquires the bacteria from diseased plants when it feeds, sucking the veins in the phloem, in the trees’ sap circulation system.

The importance of this carrier for the disease soon put into action the researchers from Esalq (the Luiz de Queiroz College of Agriculture of the University of São Paulo), more specifically Professor José Roberto Postali Parra, who had begun a Thematic Project on the insect presented to FAPESP back in 2004. This study effectively started in 2005 thanks to FAPESP financing. “Until then, the insect had not been studied in depth. The level of the population of this psyllid did not warrant research or greater control by producers. With the thematic project, we tried to learn more about the insect, to point out biological, pathogenic and behavioral measures, and to recommend using insecticides rationally, without unbalancing the environment or killing the insect’s natural enemies, like certain wasps,” explains Parra. “We found out that the insect develops better in other plants, especially in orange jessamine (Murraya paniculata), which belongs to the same family as the citrus fruit (Rutaceae) and which is used in hedges. The female insect lays its eggs on the plant’s buds. In citrus fruit, it lays an average of 160 eggs, whereas in other plants this figure can go as high as 348. “After they hatch, the nymphs emerge, to subsequently become adult insects. “We established climate and zoning parameters where the pest appears more. The greater prevalence is found in the towns of São Carlos, Bariri, Botucatu, Lins and Araraquara.”

The overwhelming expansion of the disease can be found in an experiment carried out by Marco Machado’s theme as part of another Thematic Project financed by FAPESP and begun in 2006, in conjunction with Fundecitrus. This project studies the bacteria regarding its diagnosis, biology and how to fight it. “We isolated a new orange plantation in Araraquara with ten thousand HLB-free plants, surrounded by sugarcane plantations and three kilometers away from any other orange plantation. We conducted chemical control with insecticides, with different types of applications. After three years, 15% of the plants had acquired the disease. The wind carried the insect. The situation is complex, because it could be that 99 insects got there, but that only one was carrying the disease and transmitted it,” says Machado.

FUNDECITRUSThe disease aborts seedsFUNDECITRUS

In the research field, a number of alternatives for eliminating the psyllid are being studied. “One of them is to adopt bacteria called symbionts that interfere with the insects’ behavior and biology, in addition to the fungi that can be used as control agents,” says Parra. This type of biological control is used like an industrial insecticide, by applying microscopic fungi of the Beauveria bassiana species mixed in water to the insects and plantations. The fungus is inert as far as vegetables and humans are concerned, but is a parasite in the adult insects and the nymphs, leaving them dried out, as if mummified. Professor Parra’s team is also taking into account the possibility of isolating sexual pheromones, substances secreted by the females to attract the males. These pheromones could perhaps be used in traps to eliminate the males and reduce the insect population. However, the most promising substance to halt the psyllids’ advance may be on guava trees. “The guava tree produces certain substances that repel the insect; this was first observed in Vietnam, where guava and orange trees are alternated in the same plantations,” says agronomist José Belasque Júnior, a researcher at Fundecitrus.

International studies to identify and synthesize these volatile guava substances are being conducted by INCT, the National Institute of Science and Technology of Semiochemicals in Agriculture, with financing from FAPESP and from the Ministry of Science and Technology. This institute is headquartered at Esalq and is coordinated by Professor Parra, along with the University of Pennsylvania, USA, the University of Valencia, Spain, and the Max Planck Institute, Germany. “The idea is to produce these substances in the future in the orange trees themselves through transgenetic techniques, in order to repel the insects,” explains Parra. He also includes environmental husbandry in the arsenal of weapons to fight this insect, by using the Tamarixia radiate wasp, which does not harm agriculture or man, to attack the insects’ nymphs. In studies conducted in the town of Araras, releasing this wasp in the region’s plantations resulted in the elimination of 51 to 72 percent of the insect’s nymphs. “The results are reasonable, but we need to do more lab research and studies in other regions.”

Even with all these alternatives, Professor Parra, who has been researching insects connected with agriculture for more than 40 years and who also raises insects for university studies, believes that this is a major challenge, perhaps the greatest of his career. “The insect is complicated and hard to breed, which makes us dependent on capturing it in the field. There is also the issue of the population of these insects, which varies during the course of the year, from season to season and depending on the weather, in terms of temperature and rain, without any apparent system, which has kept us from establishing models of its presence in the field,” says Parra. As part of the Thematic Project, which also has partnering agreements with Fundecitrus, IAC (Agronomical Institute), the Biological Institute and the University of California at Davis, Professor Parra’s group had found another problem: Some of the chemicals used as insecticides against the psyllid are no longer efficient, but may kill the little wasps used for biological control. Chemical control is excessive, carried out as often as twice a month. It is impossible to contain the disease just by controlling the insect; additionally, there is not enough knowledge about this type of application,” says Machado, from the citriculture center.

fundecitrusOnce infected, the plan will show symptoms within one yearfundecitrus

If the insect is complicated, the bacterium is no less so. It was only identified in 1970, in a laboratory in France. To this day it lacks a definitive taxonomic identification or a scientific name accepted worldwide. This is why it is referred to Candidatus Liberibacter. It has three species, Ca. L. asiaticus, found in larger numbers in Brazil and the cause of the deleterious infection, Ca. L. africanus, milder and not found in Brazilian plantations, and Ca. L. americanus, rarely found in Brazil, but dangerous and described in 2004 by a group of researchers from Esalq and Fundecitrus along with French researchers from INRA, the French National Institute of Agronomic Research. The identification was achieved through sequences of DNA segments. The bacterium maintains the status of candidate because researchers have been unable to grow it in laboratories, in vitro, to then isolate it. However, this situation may change, because in May of this year, a group from the USDA (United States Department of Agriculture) managed to grow it in a laboratory, according to an article in the journal Phytopathology. “One needs to find a broth that is to its liking, and this is achieved by trial and error,” says Professor Elliot Kitajima, an electronic microscopy expert at Esalq. He and Professor Francisco Tanaka obtained one of the best pictures of Liberibacter in the phloem of the ornamental plant Catharanthus roseus. “The concentration in orange trees is very low; one cannot produce images such as that obtained through the Catharanthus,” he says. “There is no relation between the concentration of bacteria and the damage to the phloem,” says Machado. Even so, the few bacteria must secrete toxins that harm the phloem’s functionality. “Quickly, just about half an hour after the insect that carries the bacteria pricks the plant, the latter becomes infected, but the evolution is slow and symptoms may not appear until one year after inoculation,” says Parra.

The struggle against the yellow dragon also involves learning about the bacterium’s genome. The USDA completed the genetic sequencing of Liberibacter asiaticus in 2008. The Asian strain of the disease has a small genome, with approximately 1.2 million pairs of bases, whereas the bacterium Xylella fastidiosa, which causes CVC (citrus variegated chlorosis) has 2.4 million pairs, and Xanthomonas axonopodis citri, the cause of citrus canker, has 4.5 million pairs. Xylella was the first plant pathogen in the world to have its genome sequenced, an experiment completed in February 2000 by researchers from São Paulo state universities and institutes financed by the FAPESP Genome Program, which also sequenced Xanthomonas. “The Liberibacter’s smaller genome means that it is even more specialized than the others,” says Machado. He also coordinates the newly created INCT (the National Institute of the Science and Technology of Genomics for Citrus Improvement), which encompasses institutes and universities from the states of São Paulo, Bahia, and Paraíba, as well as the University of Florida, USA. This US state is also the victim of greening, this disease having been identified there in 2005. Florida, which has more than 70 million orange trees, is the world’s second largest citrus producer after São Paulo, the leading citrus producing state in Brazil and the state that accounts for roughly 80% of the total fruit. Together, Florida and São Paulo account for approximately 40% of world production.

fundecitrusFrequent inspections are fundamental to halt greeningfundecitrus

However, as if two Liberibacter bacteria were not enough, in 2007 a phytoplasm (a bacterium with no cell wall) was identified in plants with greening symptoms, but no apparent Liberibacter. This was confirmed in molecular PCR tests. With the aid of the French researcher Joseph Bové, from INRA, and of Professor Kitajima, the Fundecitrus researchers announced the bad news and prepared a new test that is already in use. Studies to better understand this bacteria and its effect on citrus plants are being conducted by several groups.

The disease’s complexity demands an increasingly large number of researchers, as a third Thematic Project financed by FAPESP and begun in 2008 illustrates. “Our aims is epidemiologic: we study the disease’s dissemination as related to time, the speed with which the infection reaches plantations and the insect, and space, checking on the flight habits of the psyllid, which can be carried by wind for hundreds of meters, all of this being based on molecular analyses in the several states of the disease,” says Professor Armando Bergamin Filho, also from Esalq-USP. “One of our chief concerns is the role of the orange jessamine as the insect’s and bacterium’s host. We will look into the need to eradicate it as well,” says Bergamin, who hopes to have proposals for controlling the disease by the end of project in 2012. Bergamin emphasizes that the eradication of diseased citrus trees is a fundamental form of control. “Removing infected trees is already a federal law, but many producers prefer to only apply insecticides and cut off branches. It is useless for one producer to eradicate his trees if his neighbor doesn’t.” He believes that government inspection should also be more effective, both in terms of requiring compliance with the diseased plant eradication rule and in demanding that healthy seedlings be used, although in the State of São Paulo there is a law that requires that seedlings be purchased from nurseries protected with screens and certified, to avoid the dissemination of other diseases.

“The challenge is to persuade citrus growers that they must pull up the plant, especially among the medium-sized and small farmers, who are the majority,” says Belasque, from Fundecitrus. In São Paulo, there are over 5 thousand properties with citrus plantations. “We have a team of 21 agronomists spread throughout the state in and contact with producers, delivering lectures and monitoring the cases of the disease, which is already spreading in all of the state’s citriculture areas.” Belasque believes that the best solution would be greening-resistant citrus varieties, but this may take another two to three decades to come true. Meanwhile, the producers must comply with a series of plantation inspections every year. The Bureau of Agriculture recommends this be done three times a year, starting this year, including the mandatory issuing of reports.

The nearest hope of faster and assured inspection of diseased plants in the field lies in the electronic systems that are being developed by two groups of researchers from São Carlos. The experiments resort to the principle of fluorescence, with different procedures and techniques using the emission of light by the leaf after it is lit by a laser or a LED (light-emitting diode). This is a sequel to the study led by Professor Luís Gustavo Marcassa, from the University of São Paulo’s Institute of Physics at São Carlos, in which the researchers tried using lasers to identify citrus canker (see Pesquisa FAPESP no. 80). “We have reached a result that shows, upon analysis of the leaves, that 95% had something wrong with them as compared to a healthy leaf, whereas 65% provenly had canker,” says Marcassa. The study consists of lighting the leaf with light from an optic fiber and to capture, with another fiber, the absorption of light with reflection that has been altered by the bacterium. The data sent to a computer reflects on a graph the possibility of the plant being infected. Marcassa is conducting a similar study about greening. “Now I am not using lasers, which require a higher level of care and are more expensive, but high-powered LED of different colors. We have collected as many as 16 thousand images in which we emit a color (the frequency of an electromagnetic wave) and pick up the emission in another color,” says Marcassa. The experiment concerning greening is in its early stages and the idea is to take the equipment to the field, in the near future, or to leave it somewhere we can access it, on average, one day after collection, a time span during which the leaf does not yet reflect any changes. The diagnosis is produced in a few minutes.

The second experiment is being conducted by researcher Debora Milori, from Embrapa Instrumentação Agrícola, a subsidiary of Empresa Brasileira de Pesquisa Agropecuária, who is studying the use of laser beams to diagnose greening early. Debora and her team developed a portable device that allows one, along with other types of precision instruments, to provide a mapping of the infestation by the disease in a way that is economically viable. “At present, visual inspection can lead to errors ranging from 30 to 60 percent of the cases, including confusing this disease with others that produce similar symptoms,” says Debora. “In a laboratory, with the device calibrated for each citrus variety, we achieved a rate of 80 to 90 percent of correct answers, and the result is available in one minute. A major advantage as compared to the PCR test, which takes approximately 10 days,” she says. This study has the support of the São Carlos Center for Optics and Photonics Research, one of FAPESP’s Cepids (Research, Innovation and Dissemination Centers). Furthermore, the researcher is coordinating a research network financed by CNPq (the National Scientific and Technological Development Council) centered on biophotonics as applied to the diagnosis of greening and including as partners the University of Florida (USA), the Centro de La Papa (Peru) and the Mayor University (Chile).

The projects
Bioecology and the establishment of strategies to control Diaphorina citri Kuwayama (hemiptera: psyllidae), the vector of the bacteria that causes citrus greening (nº 04/14215-0); Type Thematic project; Coordinator José Roberto Postali Parra – USP; Investment R$ 513,245.14 and US$ 14,266.09 (FAPESP)
2. Studies of the bacterium Candidatus Liberibacter spp., causal agent of the citrus huanglongbing disease (formerly greening): diagnosis, biology and management (nº 05/00718-2); Type Thematic project; Coordinator Marcos Antonio Machado – IAC; Investment R$ 1,058,519.78 and US$ 215,009.98 (FAPESP)
3. Molecular epidemiology and integrated management of huanglongbing (Asian and American) in the State of São Paulo (nº 07/55013-9); Type Thematic project; Coordinator Armando Bergamin Filho – USP; Investment R$ 1,105,255.22 and US$ 68,824.87 (FAPESP)
4. Optics applied to agriculture and to the environment; Type Cepid (Research, Innovation and Dissemination Center); Coordinator Débora Milori – Embrapa – São Carlos Center of Optics and Photonics Research; Investment R$ 25,000.00 and US$ 40,000.00 (FAPESP)
5. Detection of citrus canker by fluorescent image in the field (nº 08/00427-6); Type Regular Research Awards; Coordinator Luís Gustavo Marcassa – USP; Investment R$ 15,582.50 and US$ 12,536. 61 (FAPESP)