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Shedding light on orange trees

In São Carlos, researchers develop a technique that uses the laser in the identification of citrus canker

The disease is devastating. The plant begins to develop lesions, the leaves fall off and the fruit takes on strange formations on the surface of its skin. When citrus canker is detected in the orchards of orange, lemon and tangerine trees, the order – upheld by law – is to eradicate the trees and to leave the location barren of these species for two years. Although the occurrence in the state of São Paulo has been lower than 1%, the problem is worrying because if one tree is attacked it is necessary to cut out close to one hundred trees in a radius of thirty meters, causing serious losses to the citrus fruit growers, who spent between 1999 and 2001, almost R$ 100 million on citrus canker.

In order to control this disease brought on by the bacterium Xanthomonas axonopodis pv. citri, the only solution is to put in place a battalion of inspectors with programmed visits into the contaminated or suspected contaminated areas. Other controlling measures, beyond eradication, have not solved the problem. The spraying of an insecticide may kill the bacterium on the external part of the plant, while in the interior, the microorganism continues to reproduce.

The inspection is carried out through sampling directly in the fruit grove – one in every five trees -, and each inspector analyzes close to one hundred and ten plants daily. But this tiring work – which is difficult to carry out, and is subject to human errors – will shortly be getting a new weapon probably in the form of a lamp, endowed with a laser beam, that will make things easier, give more precision, and mainly anticipate the diagnosis of citrus canker in the trees.

The technique – a novelty in the world – is under development in one of the branches of the Optics and Photonics Research Center (Cepof), located in the Physics Institute of the University of São Paulo (USP), in the town of São Carlos (the other branch is at the Physics Institute of the State University of Campinas – Unicamp), one of the ten Innovation and Diffusion Research Centers (Cepids) financed through FAPESP. The research also has the backing of Fundecitrus (Fund for Citrus Plant Protection), the institution maintained by the producers and the fruit juice industry.

Absorbed energy
In order to arrive at the citrus canker detector, professors Vanderlei Salvador Bagnato, Cepof’s coordinator in São Carlos, and Luis Gustavo Marcassa started from a phenomenon of physics called fluorescence spectroscopy, present in certain molecules that absorb energy and emit light after having been excited by another light source. “In the case of citrus canker, when we shine a monochromatic laser beam of green color over the orange tree, the substances present in the bacteria absorb this energy and respond with another color, or that is to say, light of another frequency”, Bagnato explains. The disease, nonetheless, cannot be verified simply with the green laser beam. One needs to use a yellow colored visor that cuts out the green frequency to the human eye. In this manner the lesions due to the disease are identified much more easily during an inspection. There is also a high probability of identifying sick trees that have not yet shown the symptoms of citrus canker. This possibility, which will be tested in the laboratory, is totally out of the question in a simple visual inspection.

Bagnato and Marcassahad had already made use of the laser and fluorescence spectroscopy for the diagnosis of various types of cancer (skin, mouth, esophagus, urinary bladder, lungs) in a project that is being developed at Cepof. In the same manner, a laser beam is cast over the region of the body to be studied and the response determines the type of tumor, if it is malignant or benign (see Pesquisa FAPESP No. 74). The beam is carried along an optical fiber, normally used in telephone transmissions, which is made up of seven segments. One sends the light, and six to collect the luminous response. Both the detection of cancers and of citrus canker make use of software especially developed for the equipment.

In the plants the comparison – done through a computer screen – allows the visualization in graphical form of the differentiated frequencies when the plant is healthy and when it is infected by the Xanthomonas. The level of fluorescence changes from that of a normal plant to an infected plant where metabolic alterations of the molecules brought about by the bacterium occur.

The equipment that will go into the fruit groves will firstly make possible a visual test by way of a laser beam and observation through special glasses. Afterwards, if some problem is detected, the affected part of the plant will be submitted to confirmation of the canker presence by software installed in a laptop linked to an optical fiber system to collect the spectrum of the leaves. The identification of other microorganisms present in citrus fruit is also included in the team’s plans. “We know that laser emissions can be the same and the responses different for each bacterium or fungus”, says Marcassa. “We are going to create a data bank for the systematic detection of diseases covering the fluorescence spectrum for each organism in order to simplify its identification”, Bagnato says. The researchers have already obtained preliminary differentiated results on other diseases, such as Citrus Variegated Chlorosis (CVC), also known as the yellowing disease, caused by the bacterium Xylella fastidiosa, and of citrus measles, a disease that as yet has not had its cause identified.

In an experiment with the canker, comparative tests were carried out, making a comparison between orange trees of the variety Pera Rio inoculated with the Xanthomonas and healthy trees. With the first data in their hands, the two researchers filed the patent for the new technique for detecting citrus canker at the National Institute of Industrial Property (Inpi) in April of this year. The Cepof researchers have certainly opened up an immense field in the construction of new equipment that might help in the diagnosis of agricultural diseases without the need for taking a sample to be analyzed in a laboratory, a procedure that greatly slows down the start of the control of diseases.

The development of the technique began some two years ago when the agronomy engineer José Belasque Júnior, from the Scientific Department of Fundecitrus, got in the touch with the Physics Institute at São Carlos. “We were looking for something to increase the efficiency of an inspection”, Belasque recalls. “We had been thinking about glasses or spraying the plant with some type of ink so that we might more easily identify the canker.”

The correct leaf
Currently the inspection is visual through a person trained by Fundecitrus. “There are trees of up to four meters in height and it is possible that only two or three leaves are already infected in the middle of some twenty to thirty thousand”, the agronomist explains. In the current procedure, all of the properties that have shown contamination are periodically inspected and, once a year, 10% of the citrus groves in the state of São Paulo are randomly chosen for inspection. On finding a leaf that is suspected to have canker, the inspector invites in the producer and a representative of the State Agriculture Secretary or Fundecitrus. Only then can the leaf be removed and sent to the laboratory of the Biological Institute, which will issue a test result.

If positive in only one plant, a thirty meter radius of plants is cut all the way around, which is equivalent to about one hundred trees. If the number of diseased examples exceeds 0.5% of the total number existing in the grove (an orchard with at the minimum two thousand trees), all of this area will be eradicated. Those that are cut must be burned on the location itself, and all of the other citrus trees in the region must be investigated. In Florida, USA, the country that is the second largest world producer of citrus fruit, behind Brazil, where the climate is more favorable to the dissemination of the disease, eradication is mandatory in a radius of 576 meters.

Loses for all
The occurrence of the disease has been falling over the last three years in the states of São Paulo and Minas Gerais, regions over which Fundecitrus acts. Last year the presence of canker reached 0.07% of the groves in São Paulo, and in Minas it has not been detected for two years. Even with this very low rate, since 1999 the eradication has represented the cutting down of close to 3.5 million plants and direct losses at the level of R$ 100 million. One can add on to this loss in the state of São Paulo, a further annual R$ 20 million for Fundecitrus resources and those of the State and federal governments with inspections and in the eradication of diseased plants.

Therefore, this bacterium that probably originated in Asia, where the disease is endemic, it is not just a ghost that only comes back to haunt, but a real enemy for the largest citrus fruit grower in the world and Brazil’s second largest agricultural industry after sugarcane. It generates some US$ 3.5 billion per year in the country and employs close to 400,000 people. Income generated through exporting annually reaches US$ 1.2 billion in juices and natural fruit. The weight of canker in Brazilian citrus growing indeed influenced FAPESP’s decision in the choice of the bacteria Xanthomonas citri as the second vegetal pathogen to have its genome sequenced after the pioneering Xylella fastidiosa.

“The incidence of yellowing is much higher, reaching 38% in the State of São Paulo, but in this case it is not necessary to cut down the tree, the disease being controlled with the pruning of the attacked branches and by combating the sharpshooter, the bacterium vector”, explains Belasque. Always in partnership with Fundecitrus, the sequencing of the Xylella involved an investment of US$ 13 million, and that of the Xanthomonas, completed in December 2000, one of R$ 2.2 million. The researchers involved in these projects are now studying the chemical interactions between each genome and the proteins synthesized by the genes.

Keeping the Brazilian citrus fruit sector competitive depends both on the research carried out in the laboratories of the Genome Project and in the development of new techniques and equipment for the detection of the diseases. Belasque believes thatin a short space of time the use of the laser will gain ground and importance in the identification of canker and of other citrus diseases.

“For example, we hope that it will be useful in the early identification of citrus black spot, a disease caused by the fungus Guignardia citricarpa.” This disease has not reached all of the regions of the state and does not involve eradication, but the symptoms only appear as small dark spots during the ripening of the fruit. “The fungus will have been there for some time, but it is not possible to detect it before the appearance of the symptoms,” Belasque explains. Fundecitrus’s intention is to also use the future equipment for the detection of canker and other diseases via the laser in the locations where seedlings are produced, a strategy that could avoid a greater dissemination of the fungi and bacteria. In the case of canker, contamination can occur via a combination of wind and rain and man himself, who can carry the Xanthomonas bacteria on his hands, clothes, shoes or even on objects.

Productive partnerships
In the opinion of Bagnato, the first prototype of equipment for use in the field should be ready in a year. “Then we will be complying with one of Cepof’s missions, which is the establishing of partnerships for the resolution of society’s and the private sector’s problems”, Bagnato says. “We would like to establish a tradition in this sector.” For example, in two years Cepof has registered eight patents concerning equipment used in medicine and in dentistry.

Besides the technical solutions, the center promotes scientific research and has been publishing around forty papers per year, in international scientific magazines, coming from a team of five professors, forty five undergraduates with scholarships, thirty six post graduates (masters and doctorates) and three post doctorates. “Something else that is important is the multidisciplinary approach to out work.” In the case of the detection of canker via laser, an agronomy engineer made contact with a light physicist and the physicists went on to learn about vegetal diseases that threaten fruit growing.

The project
Detection of Citrus Canker through Laser; Modality Research, Innovation and Diffusion Center; Coordinator Vanderlei Salvador Bagnato – Optics and Photonics Research Center(Cepof) / Physics Institute of the University of São Paulo (USP) in São Carlos; Investment R$ 50,000.00