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Virology

Without secrets

An Alellyx team presents a detailed description of a virus associated with the sudden death disease of citrus fruit trees

FUNDECITRUSFruit from the tropics: In Sao Paulo and Minas Gerais States there are around 200 million orange treesFUNDECITRUS

The first scientific article signed by a group of researchers from Alellyx Applied Genomics, the private biotechnology company, which was born through FAPESP’s Genome Program has come out. The paper was published during this month of March in the Journal of Virology setting out the genetic and molecular characteristics of a virus that the Alellyx research team considers to be a strong candidate for the agent that causes – or at the least one of the agents – the sudden death of citrus fruit trees, the disease that has already installed itself in close to two million orange trees in the states of São  Paulo and Minas Gerais. According to this study, there is a 99.7% association between the now called Citrus Sudden Death-associated Virus (CSDaV) and the illness that can kill an orange or a tangerine tree in only a few months.

Even so, this virus cannot be said truly to be responsible for the deaths of these plants. One needs to demonstrate that there is a clear and uncut relationship between cause and effect, the so called Koch’s Postulates, which consists in inoculating the supposed agent responsible for the disease in healthy organisms, in this case orange trees, and verifying whether or not they contract the disease. This is slow painstaking work, in which one needs to abide by the vagaries of the virus, in which the incubation period can stretch to three years. It is only then that the first symptoms appear: the loss of shine on the leaves and the blocking of the pores that conduct the sap from the crown to the roots. Consequently the roots die and with them the plant.

The Alellyx researchers may not have in their hands the certainty for which they yearn yet, but not even this stopped them from celebrating the publication of the paper, a historic milestone in the history of the company. The study, some ten pages in length, which came out in the Journal of Virology, a first class international magazine in the field of virology, indicates that it is possible to conciliate product developments with high quality scientific research, just as the five founders of the company intended  – all of them are specialists in molecular biology and/or bioinformatics, who did not want to abdicate scientific rigor with which they had been working at the universities and from where they had come. “Alellyx is part of the successful initiatives in genomics”, observes José Fernando Perez, FAPESP’s scientific director. “FAPESP’s Genome Program had as its objective the formation of human resources, highly qualified both for research in the academic world and in the generation of companies.”

On the paper on the virus there is no shortage of examples of consistent academic careers that have wound up in one of the rare Brazilian companies dealing with plant genomes. Among the twenty six people that put their names to the study, there are two university professors on leave, Fernando Reinach, Alellyx’s president and the executive director of Votorantim Novos Negócios, temporarily on leave from the Sao Paulo (USP), and Jesus Aparecido Ferro, who has left for a short period of time the laboratories at the São Paulo  State University (Unesp) in Jaboticabal to dedicate himself to the company in which he is a partner. Paulo Arruda has stayed on at the State University of Campinas (Unicamp), but with fewer activities than previously, while Ana Cláudia Rasera da Silva, also in demand to look after her young daughters Amanda and Mariana, aged two and five, has left USP. “There just wasn’t enough time to do everything well”, she says.

In the team, which over a period of two and a half years worked on this virus, there are also ten biologists with their doctorate degrees and a further four with their master’s degree, as well as the twelve graduate biology students. “This work was entirely funded by private initiative, but was only possible because the public university system trained these people”, comments Reinach. The group also included two very experienced virologists who acted as consultants: the Israeli Moshe Bar-Joseph, currently with the Agricultural Research Organization (ARO), in Israel, and Elliot Kitajima, from the Luiz de Queiroz Upper School of Agriculture (Esalq) of USP, one of the most respected Brazilian authorities on the virology of plants.

Limited production
It is very rare for private Brazilian companies to disclose the results of their research in scientific magazines. In a general manner, the technicians, biologists, agricultural engineers and/or veterinarians who work in companies are not prohibited from publishing their discoveries in specialized magazines, but as well they are not encouraged to spread their findings, since essentially they are in search of a product to be sold or a patent, which at the beginning requires that the information be maintained in silence.

In the databases of scientific publications production from research centers that attend to more than one company emerges more easily. This is the case of the Fund for Citrus Plant Protection (Fundecitrus), an association of citrus fruit growers and processors whose specialists have signed fifty one scientific papers (thirty three in national and eighteen in international magazines), on their own or in conjunction with other research institutions over the last five years. The team from the Sugarcane Technology Center (CTC), of Piracicaba, controlled by the Cooperative of Sugarcane, Sugar and Alcohol Producers of the State of Sao Paulo (Copersucar), published a paper in an international magazine and has participated in at least six other studies divulged in international magazines over the past three years.

Papers only by researchers from companies or a company are even rarer. In a non-exhaustive search by PubMed, a papers archive maintained by the National Institutes of Health (NIH) in the United States, besides the  papers from Alellyx , only one other Brazilian company – Natura –  comes up whose team from Natura Innovation and Products Technology signed an article. It came out last year in the Journal of Cosmetic Science and presented an alternative method for quantifying the damage to hair resulting from the continuous use of brushes. The situation does not change much when one enters into Scielo Brasil, which groups together the best scientific publications edited in Brazil. Under the name of Valleé, a company from Minas that manufactures medications for veterinary use, two articles appear, one of them produced with the help of USP and the Pasteur Institute of Sao Paulo. The company Biobrás, the only national producer of insulin, purchased in 2002 by the Danish company Novo Nordisk, has also put on-line two studies, both produced in conjunction with other research groups.

Patents
The publication of this paper on the virus is yet another step in the strategy sketched out by Reinach, who had participated in the coordination of the sequencing and analysis of bacteria genomes that causes plant diseases, funded by FAPESP, before managing to arrange close to R$ 30 million from Votorantim for setting up Alellyx in March of 2002. Since the beginning Reinach has managed his team in such as way that the discoveries would result firstly in patents, essential for the development of innovative products, and then there would be publications capable of redeeming the team’s credibility within the demands of the scientific community.

In fact, the article discloses the genome sequences of the virus that has been the object of three patents granted in September of 2003 by the American government in the names of Walter Maccheroni and Ana Claudia Rasera da Silva, the two main authors of the paper published in the Journal of Virology. These gene sequences allow for the identification of CSDaV, through types of diagnostic tests, one molecular and the other with antibodies, as well as the use of coating molecules from the virus in plants resistance to this type of sudden death disease. “As these discoveries are protected by patents”, says Reinach, “nobody else can make diagnostic tests based on these sequences for the next twenty years.”

At Alellyx,  Reinach says, “hundreds of tests per day” are already being done, mainly control the health of the plants in new orchards. “We are already attending to major fruit growers, responsible for the planting of approximately one quarter of all of the orange trees in the state of Sao Paulo”, he says. Developed initially to attend to the needs of the company’s own researchers, these diagnostic tests may even indicate where the illness should appear, since they register signals of CSDaV in the insects that transmit them – the aphids  Aphis spiraecola and Aphis gossypii. In this manner, this type of test, that other research centers are also developing, may aid preventative measures such as the elimination of infected plants before others are infected.

For Alellyx, to conquer the market for this type of test puts them on the brink of covering the investments carried out up until now in the research into this sudden death disease – around US$ 3 million – perhaps this would be a task equivalent to the identification of the virus itself. Evidently, one is not dealing with a product to be used on a large scale, in the field, as with a hoe. For the orange producers, who are always struggling to reduce production costs and to increase their selling price, sometimes it could be cheaper to pull up the supposed infected plants than to discover if they are in fact infected with the disease against which, for now, there is no remedy. But one thing is for sure: precocious diagnostic tests for sudden death disease are essential for the citrus fruit growing sector, which cultivate 200 million orange trees, employ almost 400,000 people and generate annual business to the order of US$ 4 billion, according to the survey of the Program of Business Studies of the Agro-Industrial System (Pensa) carried out by USP and concluded last year.

Slow death
Some concepts concerning the sudden death disease of citrus trees have changed since November of 2002, when the Alellyx team received their first samples of the contaminated plants and went on the hunt for the culprit agent. At the start it was suspected that they were dealing with a virus mutation of Citrus Tristeza Virus (CTV), a disease that consumed 90% of the São Paulo orange trees between 1939 and 1949. Afterwards differences sprung up and now the Alellyx team is  demonstrating that the CSDaV is a new member of the genre Marafivirus, an integrant of the Tymoviridae family, whilst the virus of the Tristeza belongs to the Closteroviridae family. However, they have still not discarded the possibility that the two of them could well be acting in conjunction as the cause of the sudden death disease.

Also, it was thought that this disease would continue to advance on the orchards at a rate of sixty kilometers per year, which happened between 2002 and 2003. Up until January of 2004, the illness had reached 1.5 million plants in twelve municipalities in the southwest of the state of Minas Gerais and a further 436,000 in eighteen municipalities in the north, northeast and center of the state of Sao Paulo. But it did not propagate itself with the speed imagined. “Sudden death disease is contained in the northern region of the state of São Paulo”, comments Marcos Machado, from the Sylvio Moreira Citrus Center, at Cordeirópolis, in the interior ofn the state of São Paulo, where they are also researching the provoking agent and the ways of controlling this sudden death disease. “There must be a very strong environmental component, such as soil, water or climate, which maintains it confined within these limits.” The speed of this advance could be reduced or accelerated also according to the concentration of orchards; the eradication of sick plants; the control of vector insects or the transportation of contaminated off-shoots, observed Waldir Cintra de Jesus Junior and Renato Beozzo Bossanezi, from Fundecitrus, and Armando Bergamin Filho, from USP, in a study published in the magazine Visão Agrícola.

Today it is known how to contain the sudden death disease: by making use of graft  – the plant upon which the species of orange tree that it is desired to be cultivated grows – resistant, although the most efficient alternatives demand extra and expensive care, namely irrigation. The current center of attention is another disease, greening or huanglongbing (HLB). Detected in March of 2004, it has already installed itself in around 400,000 orange trees in forty-six municipalities in the central region of the state of São Paulo. As it attacks the crown of the orange trees, it can only be contained by means of a radical measure: the systematic elimination of the plants, as had previously been done with citrus variegated chlorosis (CVC). Over the next few weeks a law that authorizes government sanitary inspectors to remove the contaminated trees should be passed.

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