MIGUEL BOYAYANAn esthete against plant pathogens. This is how, perhaps in a reductionist way, one could describe the work of São Paulo researcher Elliot Kitajima, who for over four decades has dedicated himself to recording images in electron microscopy of agents that attack plants, in particular viruses. Born 69 years ago, from Japanese parents, in Registro, in the Ribeira Valley, a region with a strong presence of the Japanese colony, Kitajima graduated in agronomy at the Luiz de Queiroz College of Agriculture (Esalq), in Piracicaba, in 1958. In his long career, he has worked at the Campinas Agronomic Institute (IAC) and at the University of Brasilia (UnB), where he retired after 23 years of services rendered, and he did three postdoctoral researches abroad, two in the United States and one in Holland. At the end of the last decade, he went back to Esalq, where, as a visiting professor, he continues on active service, giving courses in electron microscopy and producing beautiful and important images of the little enemies of the plants.
Some of these photos, which captive readers of this magazine will surely have seen, are snapshots of the Xylella fastidiosa bacterium, which attacks orange groves and causes citrus variegated chlorosis (CVC), popularly known as the yellowing disease. The dramatic images of Xylella in action, clogging the channels that carry the saps in the entrails of the plant, were used in the famous article published in the Nature magazine in 2000 by the network of São Paulo researchers that sequenced the pathogen’s genome. “I had a lot of luck, and the image got a lot of publicity”, Kitajima says. But this is not his only work of substance. His other important feats are usually with viruses that cause ailments in the orange tree, like leprosies and citrus tristeza. In spite of the recognition of his work, this specialist in plant virology says that the morphologists of science, like himself, are in decline. “In my area, electron microscopy, I am practically a dinosaur, a race facing extinction”, he complains, without losing his good humor. Below, the main passages from the interview granted by Kitajima to Pesquisa FAPESP:
In biology, when a virus or any other pathogen is pretty and one succeeds in making a good record of it, is this picture also worth a thousand words, as they say?
In a certain way, yes. I am suspect to talk about this, because my work is based on images. I don’t have any pretense of being a Sebastião Salgado, but I admire people like him, who try to put a bit of esthetics and dramatic quality into the images. Whenever possible, I also try to do that. In my own way. Obviously, I don’t have his sensitivity or feeling, but I always try to make the image a bit prettier, like that one of Xylella fastidiosa. I had a lot of luck, and the image was much publicized.
Do you agree that, sometimes, present-day biology seems not to give due recognition to images?
Actually, the morphologist in science is disappearing. In the old days, when this morphological part was more in vogue, I would open a [scientific] magazine and recognize by the kind of image who its author was. The disdain for the morphological vision has resulted in mediocre illustrations in the publications. With good experience, it is possible, in my area, to distinguish, in the midst of cell organelles, in a section of the infected cell, the presumable particles of the viruses, for bacterial or fungal cells. Of course, there is any abyss between being and seeming. But in these cases we can resort to more sophisticated techniques, to be sure of the identification. In electron microscopy, I’m practically a dinosaur, a race faced by extinction. The undergraduate student today only wants to do molecular biology and forgets the fundamentals. It’s true that molecular biology and biotechnology are rendering inestimable services to humanity. However, I like to say that if I give him a bit of nucleic acid, the pupil does wonders, but he doesn’t know anything about the organism that generated this DNA or RNA. Which means to say that they lack this more holistic vision. I have never worked with molecular techniques, but I have the conviction that, if it were necessary, I would do it one day. Molecular biology is usually a series of recipes. If you have the reagents, the thing works. Even a good undergraduate student does sequencing. I get worried with people that just learn the routine. They do not learn to think about the whys and wherefores of their acts and to create. Fortunately, a confluence between molecular and morphological techniques is happening, so that it has been possible to visualize molecules in action in the insides of the cell.
How did the opportunity arise to work on the Xylella?
Although I had worked in the 1970’s with a Xylella that attacks plum trees, in collaboration with an Argentinean group, I had never worked with the CVC one. But I ended up getting involved because of one of my children [João Paulo Kitajima, who, at the time, was in bioinformatics at Unicamp, and is working today in the biotechnology company, Alellyx]. He was setting up the project’s website and said “hey, Dad, get me a good image of Xylella”. I sent a few, he liked the one that illustrated the project’s website and ended up coming out in Nature, and the rest is history. The image really came out very beautiful, dramatic. That’s the reason why I got so involved. But it was something marginal to my work.
Do you know how many images of plant pathogens you have now done with electron microscopy?
It’s difficult to do an estimate. There have certainly been thousands, but I have never been sufficiently organized to keep a decent portfolio. Besides this, as I worked at different institutions, here and abroad, the majority of the negatives was archived in these places. Today, with digitalization, the task has become simpler. The majority of those we can regard as the best are to be found in the scientific articles that I have published. I would say that some of my small glories were to see some of them reproduced on the covers of scientific magazines.
Why did you get interested in plant virology, and, more specifically, in electron microscopy?
I was hired by the IAC in 1959 to do electron microscopy on plant viruses. Why is the electron microscope important for virology? Unlike funguses and bacteria, viruses cannot be seen in the ordinary microscope. Sometimes, we see a virus aggregate in the ordinary microscope, but we cannot manage to visualize each virus individually. Virology has always been a pit of ignorance. When they didn’t manage to isolate the agent that was causing a disease in a plant or animal, the scapegoat were the viruses. Many diseases that we know today to be caused by bacteria, by phytoplasms/spiroplasmas [bacteria without a cell wall] or by viroids [pathogens that are smaller than a virus, made up of pieces of RNA] were regarded as being of viral origin. Gradually, man has cleared up these misunderstandings. Save for rare exceptions, plant viroses do not destroy crops extensively, although they can cause small but persistent losses, which end up being ignored. Hence the reason for farmers and laymen not knowing the importance of plant virology.
Could you cite a relevant case of losses or extra costs caused by a virus in agriculture?
Citrus leprosis, for example, is one of the most important diseases to attack the groves. In the state of São Paulo, it hasn’t caused major damage to the orange groves, but this is due to investments in the order of US$80 million a year in acaricides, to control the Brevipalpus phoenicis mite, the vector that transmits the virus of the disease to the plant. Controlling the mite, the disease is controlled. If the fruit grower does not do this, the cost for him (and for society) is going to be much greater. Incidentally, my Thematic Project at FAPESP is based on the group of viruses to which the leprosis virus belongs.
Is the resolution of a present-day electron microscope much greater than when you started your work, over 40 years ago?
From then until now, not much has changed in terms of resolution. What has changed is the ease of operation. In the old devices, the adjustments were manual. In today’s, everything is automatic and computerized. The older microscopes were designed for big, long-armed Americans. I am short and suffered a lot to operate this equipment. But from insisting so much, I learnt to get round these difficulties. At the beginning of my career, around 1961, we received the first electron microscope used in agronomic research at the IAC. As we did not have many resources, the majority of times I myself would do the maintenance of the instrument. I learnt with the technician from the company how to assemble and disassemble it. The electronic part was far simpler, since they were still using valves. It was easy to know when there was a problem. You just had to look for the burnt-out valve and replace it. The complexity of today’s apparatuses calls for knowledge of electronics. That is why I do not dare any more to try to do repairs.
What are your most important works in the area of electron microscopy?
My first relevant achievement was in 1963 or 1964, when we were pioneers in visualizing the citrus tristeza virus and publishing an article on the pathogen. I mean, it is even possible that someone may have seen the virus before us, but we were the first to associate the long particles found in extracts from sick plants to the pathogen. It was my only article in Nature, in actual fact, a small note. Tristeza almost did away with orange farming in the state of São Paulo in the 1940’s, until it was discovered that you just had to replace the orange tree’s rootstock with kinds that tolerated the virus. Later on, a “vaccination” program using milder forms of the virus to avoid infection by the severe forms was successful and kept the groves free from the problems of tristeza. By the way, in this story of “pre-immunization”, Doctor Álvaro Santos Costa [one of the fathers of plant virology in Latin America and an eminent researcher at the IAC, who died in 1998] played an important role. He discovered that, if we inoculated the orange tree with a weak strain of the tristeza virus, the strong race, which was causing the problem, would not infect the plant. It’s as if you have a cold instead of catching flu, because the cold prevents the flu virus from multiplying. Santos Costa was my intellectual father, and I owe my whole scientific and professional career to his competence, character and exacting nature. I would like to mention other scientists who had an influence on my career, like Adolpho Brunner Jr., who, with enormous patience and competence, started me off in electron microscopy at the Butantan Institute, and Wladimir Lobato Paraense, at UnB, a moral and intellectual guide.
You did three postdoctoral attachments abroad, which, I imagine, must have yielded interesting work.
I stayed at the University of Chicago from 1968 to 1969. Although the institution was fantastic, we didn’t appreciate the city much. We had a complicated year. My work was centered on cytochemistry at the Cell Biology Institute, under the guidance of Hewson Swift, where there was nothing about phytopathology or plant viruses. Luckily, two hours from there, in Urbana, at the University of Illinois, there was an excellent plant virology group run by Dick Peters, a Dutch virologist who was also doing postdoctoral studies. He was working with a rhabdovirus, the SYVV, but was not managing to get good images of his material. One day he asked me to examine the material in the electron microscope in Chicago, and then anti-Murphy’s Law came in. I took one of the best photographs of my life. The results were published in the Virology magazine, in an article of which I was a coauthor. Since then, these images have been reproduced several times.
What exactly was this virus?
It is a virus without economic importance, which infects a weed called the sowthistle, but it had academic interest. Like other rhabdoviruses, the SYVV has a peculiar, elegant structure, and it was one of the first to be purified using plant tissue. As a consequence of this work, Dick and I became friends and we carried on our cooperation. He became a specialist in a group of plant viruses called tospoviruses, which in tomatoes, bell peppers, tobacco and lettuces cause a disease called wilt. I even did a postdoctoral attachment with his group at the University of Wageningen, in 1989 and 1990. It was probably the most productive scientific phase of my career, since I was the coauthor of 12 articles in nine months!
What other studies of yours would you highlight?
In 1972, with the help of electron microscopy, we saw in orange tree tissue particles similar to those of the citrus leprosis virus. We published an article about this and helped to reinforce the theory that it really was a virus that caused this disease. This was, in fact, an important work, since for a long time it was thought that leprosis could be caused by a fungus, and, afterwards, by a toxin in an acarid. The first to have evidence that leprosis really was caused by a virus was an American, following grafting tests, and our work contributed towards confirming this theory.
What are the challenges for Brazil in the area of phytosanitation?
There are certainly various, such as forming teams that are competent and ready to solve the existing problems and to face future challenges. There is a need for technicians on all the fronts, even people in the field. We have a reasonable contingent of researchers, but there are deficient areas. Research institutions like Embrapa and similar ones at a state level have made important contributions, in cooperation with universities, in particular the official ones, but they could have been more efficient. One feels a lack of strategic planning, a thinking group that that can help to establish the actions of the ministry and of the state secretariats for agriculture, the financing agents, the farmers’ associations and the research institutions. Another important point is contributing not only to improve productivity, but to do this without attacking the environment. We have to fight against indiscriminate deforestation and the abusive use of agrochemicals. Developing biological control technologies, aimed at our needs, seems to me an excellent route. From bodies like Ibama, one expects greater rigor, but also common sense. The difficulties lodged by this agency, for example, in the case of spotted fever, due to the existence on Esalq’s campus of capybaras with the tick that transmits the disease, are delaying the taking of obvious control measures, such as the management of these rodents. This posture is putting at risk the whole community of the campus and of the city.
Talking specifically about plant pathogens, are there emergent viruses, bacteria or funguses that may pose great threats to farm crops?
There is a coconut tree disease, the lethal yellowing, caused by a phytoplasm, which is causing serious problems in the Caribbean, in Central America, and may reach us. In Brazil’s banana plantations, there is now the danger of the Mycosphaerella fijiensis fungus, which causes black sigatoka. Asian rust has been causing damage in the cultivation of soybeans, and its presence in the crops is going to increase production costs. It is easy to control, but expensive. There are other examples. Plum trees will have to face a virosis called plum pox that has already reached Chile and Argentina, coming from Europe, and it will probably come here. There is a new disease, of a probable viral etiology, attacking melons in Rio Grande do Norte, the yellowing. The plants affected have fruit that is improper for export. Recently, in one of our citrus groves, greening was detected, a disease caused by a bacterium. It could potentially cause our citriculture industry enormous losses. Viroses in tomato plants, transmitted by whiteflies, the so-called begomoviruses, are also causing concern.
Does globalization, which has shortened the distances between people, not favor the dissemination of new diseases for the agricultural crops of all the corners of the world?
Certainly. Many of the diseases that we have today are brought by the farmers themselves, who travel abroad and bring back, hidden in their baggage, seeds or vegetable matter, often with the pathogens. Although there is legislation and some inspection, there is rarely any strict surveillance in Brazil. Even in the United States, with all the fear of bioterrorism, I have never been searched. Moreover, with the enormous number of travelers, it is practically impossible to do individualized inspections. Even in inspected substances, the shortcomings in the detection methods and the chronic shortage of personnel and resources leave enormous gaps for pests and pathogens to come into our territory. Add to this greed and the traditional “Gerson’s law” and we have all the ingredients for importing problems.
What disease has come into the country like that, for example?
In the 1960’s, citrus canker was brought from the East in material introduced illegally. Amongst orchids, there is a disease, caused by the virus of the orchid fleck, which has been distributed all over world. It is easy to understand why this has happened: collectors and traders keep up an intense interchange of live material, legally or illegally, and end up disseminating the virus and the vector. Fortunately, it is a pathogen without much importance. Its damage is cosmetic, it causes patches on the leaves of the plant. But it serves well to illustrate how pathogens and pests can nowadays be rapidly disseminated in all the parts of the world.
