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FAPESP History I

A laboratory at an orange plantation

From the pioneering studies of citrus canker to the genome of citrus variegated chlorosis, FAPESP has invested in the research that has made Brazilian citriculture competitive

CRISTIANO MASCAROPlantations in inner-state São Paulo: competitivenessCRISTIANO MASCARO

The diseases that affect citriculture in São Paulo state have been the target of successive research projects financed by FAPESP since the creation of the Foundation almost 50 years ago. From the studies of citrus canker conducted by the Biological Institute in the 1960s to the genetic sequencing of the pathogens that affect orange plantations, such as Xylella fastidiosa and Xanthomonas citri, FAPESP resources and the curiosity of hundreds of researchers have been marshaled to drive basic science on citriculture forward, fight pests and improve varieties. “São Paulo has a favorable climate, fertile soil and industry that has learned how to be extremely competitive, but we live with the greatest plant health problems that can affect citrus trees in the world,” says Antonio Juliano Ayres, manager of the Scientific Department at Fundecitrus, the Citriculture Defense Fund, an entity linked to the juice producers and industry, which has invested R$9 million in research and maintains important partnership agreements with FAPESP. “Citriculture in São Paulo state wouldn’t be as strong were it not for the ongoing investment in science and technology in this sector – and FAPESP played a fundamental role in this,” he states. This economic activity turns over US$2 billion a year in the state and generates 400 thousand direct jobs. São Paulo has the world’s largest plantations of oranges for juice (627 thousand hectares).

The scientist Victoria Rossetti (1917-2010), a researcher at the Biological Institute, was a key name working on this line of research. The first woman ever to graduate in agronomical engineering from Esalq (the Luis de Queiroz College of Agriculture) at the University of São Paulo, Victoria applied, in 1963, for the Foundation’s first grant for the study of the control of citrus canker, at a time when the disease was spreading and dramatically threatening the state’s orange agribusiness. One of the most serious orange plantation diseases, citrus canker is caused by a bacterium that attacks branches, leaves and fruit. It first appeared in Brazil in 1957, in the Presidente Prudente region. At that time, there was a lack of basic information on its forms of contagion and methods of control. The first symptom is the appearance of yellowish stains that, as the disease evolves, darken and become protuberant. There is no treatment for the disease – the solution is to eradicate the plant and part of the infected plantation. “There weren’t enough resources for the research and she was highly concerned about getting support to publish her findings and to send researchers on internships or to events abroad,” states Eduardo Feichtenberger, a researcher at the Biological Institute who worked with Victoria Rossetti.

Between 1953 and 1985, the scientist received 14 grants from FAPESP for research projects, as well as grants for researchers and support to present research at international meetings, to visit other research centers and for scientific publications. Her studies made it possible to develop a work program that is fundamental both for eradicating and for controlling citrus canker. A reference work with an analysis of the entire bibliography then known on citrus canker was released by Victoria in 1982 sponsored by FAPESP.

CRISTIANO MASCAROAn ailing orange tree: science backed public policyCRISTIANO MASCARO

While the studies conducted at the Biological Institute helped to reveal the characteristics of Xanthomonas citri, the Campinas Agronomical Institute (IAC) invested in the development of varieties that resisted and tolerated the disease better. The accrued knowledge on the disease, which previously had only been detected in Japan, led to a decision that, paradoxically, made field research unfeasible. In the early 1970s, a law was enacted according to which, to control the disease, all plants affected by citrus canker had to be eradicated. “Only in the area of Presidente Prudente was it possible to conduct research. Later, the Biological Institute stopped working on citrus canker in that region and research ended up being eradicated along with the plants,” says Marcos Antonio Machado, the director of the Sylvio Moreira Citriculture Center, a research unit connected with IAC. From the economic point of view, the decision was correct. The disease was suppressed, though it reemerged even more seriously after the arrival of the citrus leafminer in 1996 – the pest peaked in São Paulo state in 1999 and last year.

After the restriction dictated by the law, citrus canker research was resumed in the late 1990s, with emphasis on improving the methods of preventing and eradicating the disease, but, above all, with emphasis on genomics. In late 2000, 11 sequencing laboratories financed by FAPESP, spread throughout the state’s universities (USP, Unicamp, Unesp) and research institutes completed the sequencing of the Xanthomonas citri genome. Given the speed with which this was accomplished back then (just 14 months), the effort illustrated the increasing maturity of the work methods and the team – largely the same network of 192 researchers that had taken part, in 2000, in the sequencing of the genome of another bacteria, Xylella fastidiosa, which causes variegated chlorosis, another major orange plantation disease. The Xylella genome, the first full sequencing of the DNA of a pathogen in the world, became the cover feature of the journal Nature in June 2000 resulting in greater visibility for Brazilian research around the world. In 2010, Nature recalled the tenth anniversary of this achievement in an editorial.

Virtual network
In 2002, the researchers from the laboratory network indicated ways to fight the Xanthomonas citri starting with an analysis of 100 of its genes, in a study conducted in comparison with its cousin, Xanthomonas campestris. The bacteria sequencing projects were conducted under the FAPESP Genome Program.

Marcos Machado notes that one of the objectives of the Genome Project was met, i.e., training teams in the fields of molecular biology and genomics. “Thanks to FAPESP’s action, it was possible to bring together groups that would never have worked together otherwise,” he says. Researchers from several institutions and from different disciplines, from biology to medicine, including bioinformatics, then an almost unknown science, worked together in a large virtual network that brought together, as many as 35 laboratories, to fulfill a joint objective: the genetic sequencing of several organisms. However, the results of the sequencing of Xylella and Xanthomonas were different. “Sadly, there was no significant expansion in the number of studies about Xanthomonas, contrary to Xylella, which, based on the genome information, became the eighth most studied bacterium in the world,” says Machado. He highlights the fast progress of knowledge about this bacterium that was made feasible by the program. “I am unaware of any object of research that evolved as much as Xylella in terms of short-term knowledge. Even though control of the disease did not ensue, we have made a fair amount of progress, when it comes to knowledge about its pathogenicity. In 20 years, it was transformed from an unknown into a notorious bacterium. São Paulo and California have the soundest groups working on Xylella.”

Machado highlights that one of the outcomes of the research was the development by his group, of a technology that keeps the bacterium from disseminating in the plant. “We have already filed a patent request in Brazil and now we’re working on patenting this abroad.” The choice of Xylella as the first pathogen to have its genome sequenced was based on a number of the characteristics of the bacteria: it has a small genome, the mapping of which was feasible from the technical point of view, besides being an emerging problem for São Paulo citriculture. “Xyllela combined several appealing features,” states Machado. Victoria Rossetti, once again, played a fundamental role in determining the cause of CVC, which was how she named the disease in 1987, when she identified it in northern São Paulo state. “Suspecting, at first, that is was the redoubtable disease huanglongbing (HLB), or greening, she sent materials collected from the diseased plants for diagnosis to the laboratory of the University of Bordeaux, in France,” says Eduardo Feichtenberger. It was the collaboration of Victoria, Monique Garnier and Joseph Bové, from France, that enabled the French group to establish a causal relationship between CVC and the bacterium, named fastidiosa because it grows slowly. The French group supplied the Xyllela isolate to the Citriculture Center group, which prepared enough DNA to sequence.

Biological Institute collection Victoria Rossetti, in the 1970sBiological Institute collection

Loss of leaves
Research studies sponsored by FAPESP also helped to fight several other citrus diseases. In the last few years, there have been various thematic projects, that brought together several research teams to conduct investigations that can take as long as five years, involving several diseases. These include citrus tristeza virus (CTV), a viral disease that decimated more than 80% of the orange trees in inner-state São Paulo in the 1940s; huanglongbing (greening), which causes colossal damage, making the fruit and leaves drop off and drastically reducing the economic life of the affected plants; citrus black spot and postbloom fruit drop (PFC), caused by fungi; and leprosis, one of the oldest orange plantation blights. In the late 1950’s, research in which Victoria Rossetti took part, at the Biological Institute, confirmed the association between the leprosis virus and the Brevipalpus phoenicis acarus. Later, together with Elliot Kitajima, a researcher from Esalq and other collaborators, proof was found that leprosis is a disease caused by a virus and that in Brazil the carrier of this disease was the acarus B. phoenicis. “Only in the 1990s, with the advent of molecular biology and large projects, was it possible to obtain final proof of the etiology of the disease, acknowledging that the pathogen was an entirely new virus, whose classification as CiLV (Citrus leprosies virus) was internationally accepted,” says Machado. In the past, the procedure was to spray the plantations whenever the acarus was seen. Today, using pesticides is only justifiable when there is a certain amount of acari and when one can prove that they are actually contaminated by the leprosis virus. Florida was where the disease first appeared, in the early twentieth century, but it was eradicated there. Years later, probably as a result of materials smuggled into the country, the disease appeared in Brazil.

Created in 2009, one of the 44 National Institutes of Science and Technology sponsored by FAPESP and the National Council for Scientific and Technological Development (CNPq) in São Paulo has been focusing on improving citruses using genomics. Under the coordination of Marcos Machado, the National Institute of Science and Technology of Genomics for Citrus Improvement (INCT Citros) is working on three platforms, “One of them is genomic information; here the focus is on comparative and functional genome studies, which allow us to expand the information base, including the full genome, associated with the International Citrus Genomics Consortium. One of the targets is genetic transformation in order to produce genetically modified plants based on genome information,” states Machado. The second platform is based on studies of the interactions between the plants and the pathogens, with a view to expanding the base of knowledge potentially applicable in the subsequent stages of the program; and the third one centers on genetic improvement through traditional means or genetic manipulation. “We have new varieties that tolerate CVC and leprosis, among other diseases,” says Machado. The INCT efforts, which are continuing to pursue the work started by one of the Millennium Institutes that existed from 2002 to 2005, provide a sample of the research topics on citriculture that are to mobilize researchers in the future.