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Another suspect in the sights

New species of virus may be the cause of citrus sudden death

EDUARDO CESARInfected orange tree and Alellyx’s laboratory: 90% of the sick plants had the CSDV virusEDUARDO CESAR

A new species of virus may be the cause of citrus sudden death (CSD), a disease with a mysterious origin that, after its first symptoms have appeared, annihilates an orange tree in a few weeks and currently poses the greatest threat to citrus growing in the state of São Paulo and south of Minas Gerais. The announcement was made last month by researchers from Alellyx Applied Genomics, a biotechnology company based in Campinas and associated with the Votorantim group, who have identified a hitherto unknown virus, from the Tymoviridae family, in plants with sudden death symptoms.The company itself admits that it is not absolutely sure that the disease is associated with the presence of this virus, whose small genome of 7,000 base pairs and six genes was sequenced in the company’s laboratories. The scientists did not hesitate in baptizing the virus as, literally, the Citrus Sudden Death Virus (CSDV) and have now asked for a patent in the United States over the use of its genetic sequences for developing forms of preventing, diagnosing and treating the disease. If it is granted, the patent will also be valid for Brazil. “We have developed a test that makes possible an early diagnosis of the disease”, guarantees Fernando Reinach, the interim president at Alellyx.

The efficiency of the examination in detecting the presence of the new virus in an orange tree is in the order of 90%, according to Reinach.Identified for the first time a little more than two years ago, in groves in the municipality of Comendador Gomes, in Minas Gerais, citrus sudden death is, at the moment, a disease found only in Brazil. The last survey carried out by the Fund for Citrus Plant Protection (Fundecitrus), a private institution funded by orange growers and juice processors, indicates that its area of incidence covers 23 municipalities – 11 in the south of Minas Gerais and 12 in the north of São Paulo. In spite of less than 1% of the 200 million orange trees in the south of Minas and in São Paulo showing the disease, the rates of expansion and lethality of CSD are frightening. It is estimated that the disease has already caused losses of at least US$ 20 million to the sector, which employs 400,000 persons and generates exports in the order of US$ 1.3 billion.

Sudden death affects the roots of the orange trees and deprives it of its nutrients, causing a sort of heart attack. It is usually diagnosed too late. As it is not known for sure what the causal agent of the disease is, it is only possible to discover that the tree is infected after the appearance of the symptoms of sudden death, such as the loss of the shine on its leaves. It may sometimes take up to two years for these clinical manifestations to materialize in the plant. The problem is that, at this stage, the orange tree is now condemned and it is impossible to save it (at the moment, there is no cure or treatment for the disease). Hence the interest of Alellyx – and of public research institutes – to develop a test to make an early diagnosis of sudden death possible.

For the biotechnology company, one of the pieces of evidence that indicate a strong association between the new virus and the occurrence of the disease is an experiment carried out on 110 orange trees (53 with sudden death and 57 healthy ones). At the end of the study, the researchers from Alellyx found that about 90% of the trees that showed signs of the disease had the CSDV, and more or less this same percentage of the healthy plants did not have the CSDV. Another indicator of the possible pathogenicity of the recently discovered microorganism is the fact that the other viruses of the Tymoviridae family attack plants (corn, grass, oats and grapes). In the case of grapes, one of these viruses causes a problem in the roots of the plant, which shows some similarity with citrus sudden death.

These details, on their own, are the smoking gun that proves definitively that the CSDV really is the cause of the disease that is endangering citriculture in São Paulo. Alellyx itself recognizes this, although it is betting the greater part of its chips on the new virus. Confirmation that this hypothesis is correct will only come when the CSDV is inoculated in healthy plants and the orange trees develop the disease. “We have now done this and expect to get the first results eight months from now”, says Reinach. “Only then are we going to be absolutely certain that it is the new virus that causes sudden death.”

While in doubt, Alellyx itself, like other public research institutions in the citrus area, has not abandoned the idea that, before the CSDV arose, used to be the most accepted one for explaining the origin of the disease. According to this theory, sudden death has its origins in new mutations of the tristeza citrus virus, which is endemic in orange plantations today. Tristeza is a disease that almost wiped out the orange plantations of São Paulo in the 1940’s. Besides its discovery of the CSDV, Alellyx found mutations in eight regions of the genome of the tristeza virus that may be related to the occurrence of sudden death. But the company thinks that the evidence in this direction is more fragile than the evidence that sustains the thesis that the new virus, the CSDV, is the agent of sudden death. Be that as it may, Alellyx has also asked for a patent over the use of the regions identified in the tristeza virus.

Alellyx’s wager that the new virus has to be the cause of sudden death was received with reservations by specialists from the citrus sector. “The company’s study opens up a new front for investigation, but it still does not prove anything”, explains Nelson Gimenes Fernandes, a researcher and Fundecitrus’s executive secretary. “More work, in greater detail, will have to done before arriving at any conclusion.” For Fernandes, the greatest proof of this is that Alellyx itself, in spite of having found the CSDV, has not yet discarded the hypothesis that CSD may have its origins in genetic alterations in the tristeza virus. Or even that both the viruses may act together to unleash the disease. Agronomist engineer Marcos Antônio Machado, from the Sylvio Moreira Citrus Center, also regards the details divulged by the biotechnology company as inconclusive.

“There are over 20 latent viruses in citrus plants, which may or may not cause the plant problems”, ponders Machado, who is also studying the origin of the sudden death. “The sample of 110 trees used in the study by the company is too small to establish an association beteen the disease and the new virus.” Progress in the researches into the new threat that overshadows the orange groves will say whether or not the CSDV is behind the sudden death.For FAPESP’s scientific director, José Fernando Perez, the results attained by Alellyx in its work on CSD, even though they are not yet entirely conclusive, do bear witness that it is possible to do high quality science in Brazil in the business environment.

“In particular, research focused on the area of technology, which has the objective of solving problems”, comments Perez. The announcement of the discovery of the new virus, which may be the causal agent of the sudden death, occurs six years after the launch of the first Brazilian venture in the area of genome sequencing, the Xylella fastidiosa bacterium project. Xylella also causes a disease in citrus fruits, Citrus Variegated Chlorosis, the yellowing disease. The researchers who founded Alellyx are veterans of that pioneer enterprise, which was financed by FAPESP.