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João Lúcio de Azevedo

João Lúcio de Azevedo: Unjustified fear

Expert on plant microorganisms says only 1% of fungi and bacteria cause problems

Eduardo CesarIt is odd to hear geneticist João Lúcio de Azevedo talk about his work. A strong advocate of plant fungi and bacteria, he pokes fun at people’s ignorance when they appear worried about coexisting with them. “Only one percent of microorganisms give rise to problems; all others are benign, where plants are concerned,” he explains, whenever he has the opportunity to do so. The last time this happened was at the FAPESP auditorium, where a seminar was held on the challenges of tropical agriculture, as part of the activities of the 2009 Bunge Foundation Award. Azevedo won the prize in the Life and Work category, while Carlos Eduardo Pellegrino Cerri won it in the Youth category. Both are from ESALQ, i.e., the Luiz de Queiroz College of Agriculture of the University of São Paulo (ESALQ-USP).

At the age of 72, João Lúcio de Azevedo’s peers perhaps regard him as the Brazilian researcher with the greatest knowledge of the genetics of agricultural microorganisms. His is also a professor dedicated to the task of training new researchers. Up until September, he had been the advisor for 98 masters and 68 PhDs, from Manaus in the state of Amazonas, all the way to Caxias do Sul, in the state of Rio Grande do Sul. An astonishing number, which he explains pragmatically: it is in the post-graduate courses that the specialized personnel required for research is to be found. Therefore, that is where one must cultivate and pluck the best researchers.

Azevedo’s base has always been ESALQ, although he did two post-doctorates abroad. However, he frequented and set up centers of genetics at the universities of Campinas (Unicamp), Caxias do Sul (UCS), Goiás (UFG), Brasília (UnB) and Mogi das Cruzes (UMC). At present, he is microbiology coordinator for the Amazonian Biotechnology Center in Manaus. For almost 11 years, he was the joint coordinator of Agronomy and Veterinary Medicine at FAPESP’s Science Office. He has written a book that became a benchmark reference in the field, Genética de microorganismos [The Genetics of Microorganisms] (Federal University of Goiás, 1998), whose second expanded and revised edition was published recently. His studies have yielded two patents, one in association with Fundecitrus and the other, still under way, with Companhia Suzano de Papel and Celulose, a pulp and paper company. Separated, with two children and two grandchildren, João Lúcio de Azevedo, who is invariably in a good mood, talked to Pesquisa FAPESP about the poorly understood contribution of microorganisms to agriculture.

During your presentation at a seminar at FAPESP you mentioned that plants are mere substrates so that microorganisms can live. This view is similar to the one that some biologists hold regarding humans. What led you to this conclusion?
Observation. The biologists are right. There are more microorganisms in the human body than human cells. The same thing happens with plants. The plant’s cell is a lot smaller, but if we count the number of microorganism cells and plant cells there are more of the former than of the latter. There are almost 100,000 bacteria per gram of plant.

Are these bacteria that just live on the plant itself?
Some are characteristic of the plant, but have the capacity to live and to reproduce in the soil as well. This was discovered by a German, De Bary, in 1866. He discovered that there were bacteria on the plant, but he unfortunately said they were neither beneficial nor harmful. Only in the 70’s did the Swiss and Americans notice that they were much more beneficial to plants than harmful.

Are these bacteria that just live on the plant itself?
Yes. It was in the second half of the twentieth century that the early work with endophytic microorganisms first appeared – “endo” from within and “phytic” from plant. These are the fungi and bacteria that act within the plant. The discovery in the late 70’s showed that these microorganisms are extremely important. They make phosphate soluble: the plant needs phosphate and they take this substance out of the soil and feed it. In turn, the plant supplies the microorganism with a substrate for it to grow. Many of them fix nitrogen from the air, others make antibiotics that kill pathogenic microorganisms and there are those that produce toxins to kill invading insects. Some kill herbivorous animals, such as goats and cattle. It’s what we wrongly call a poisonous plant. But it’s not at all poisonous; what kills is the fungus it carries and not the plant itself.

It’s not a substance of the plant itself that kills?
Most of the time, it’s not. Obviously, there are poisonous plants. But many of these pasture plants, that cattle eat causing them to die, carry a fungus that is the true cause of death. It lives within the plant and protects it. It’s great for the plant… But there are others that protect against drought, for example.

How can a bacterium protect the plant against drought?
It’s a type of microorganism that comes from the roots and spreads to places where it manages to find some moisture, which it takes to the plant. It’s what we call protection against hydric stress. There’s another type that produces an antibiotic to kill plant pathogens. Some of these antibiotics might also be used in humans.

So, in fact, there’s potential in these microorganisms for human health, right?
Enormous potential. It’s common to hear that some plants are medicinal. Many are, but many are not. It might be that the microorganism that lives within the plant produces the curative substance. It’s also amusing to hear that the same plant may be good in Manaus and bad in Recife. It’s the same plant. It may be that one has the microorganism and the other not.

Give an example of a plant microorganism that has been transformed into a medicine.
One of them is taxol, used to treat breast and uterine cancers. It’s produced by the Taxomyces andreanae fungus that lives on various plants and that was discovered in the United States. It was interesting, because they were destroying all plants that produce taxol and they take a long time to grow. But then they took the fungus, fermented it and managed to produce more taxol than the plant. From the biotechnology point of view, it’s a great step forward. This has all happened since 1980.

But the work of Johanna Döbereiner on nitrogen- fixing bacteria comes from the 70’s, before this period.
That’s right. Professor Johanna worked at Embrapa, in the same line of research with plant microorganisms. At the time, although they were endophytic, they were called atmospheric nitrogen fixers, some of which could produce nodules on plants and others not; the latter were called diazotrophic. She was a pioneer in this area and discovered this in the tropics, even though she was of Czech origin, but living in Brazil.

Is it a fact that nitrogen-fixing bacteria were one of the reasons for the success of agriculture in Brazil’s Cerrado [savannah] region?
It was one of the reasons. But undoubtedly the use of bacteria played a major role.

What’s the story of the growth of eucalyptus stimulated by microorganisms?
“In the FAPESP seminar I showed a very small eucalyptus tree with no endophytic bacteria and another big eucalyptus that had them. Suzano is using bacteria – including the Stenotrophomonas multifolia – to inoculate eucalyptus saplings and help them grow more. This is another example of how microorganisms help plants. Nevertheless, if we leave the sapling in the ground without microorganisms they enter later because the plant defends itself and looks for what’s best for it. The technique is interesting in the first 30 days of the plant’s life. This work was done by my group and is being patented.

EDUARDO CESARWill we ever reach the point where, by using just microorganisms, we will manage to eliminate the large amount of chemical additives used in the soil?
Probably not. It’s difficult because agriculture is very artificial. The Amazon Forest, for example, has few pests. There’s a natural equilibrium in that environment. Now, if we plant hundreds of hectares with just oranges, at some point an imbalance will occur. Microorganisms and insects will appear that caused no problems before. That’s what happened with Xylella fastidiosa. I don’t believe that this bacterium was introduced into our orange groves accidentally. It probably already existed here, but because of the natural imbalance caused by an immense quantity of the same crop it began to proliferate and caused citrus variegated chlorosis – amarelinho.”

Is it possible to estimate the number of microorganism species that exist?
Worldwide, it is estimated that there are 1.5 million species of fungi and 100,000 species of bacteria. Of these, we know between 70,000 and 80,000 fungi and 5,000 bacteria.

It seems little…
The estimate was made in England and is not accurate. I believe there are more, too, but these are the figures we have. One of the criteria researchers use is that there are more than 300,000 species of known plants. Every time we work with a known species, another four or five new microorganisms appear. You multiply 300,000 by 5 and you get 1.5 million fungi.

What’s the estimate of the number of fungi in the Amazon?
In Brazil we have more or less 60,000 plant species, most of them in the Amazon. Multiplying 60,000 by 5 will yield some 300,000 species of fungus. There are many things to be discovered. The funny thing is that whenever people talk about fungi the comment is negative. Now, 99% of these 300,000 are likely to serve for good things and only 1% causes some type of problem.

In the August issue we interviewed virologist Edison Durigon, from USP, who has a project for capturing migratory birds in the Amazon to monitor foreign viruses entering Brazil. He says that it’s essential to have good safety infrastructure in order not to catch a dangerous virus in the middle of the forest and unwittingly spread it to a major center. How is it with plant bacteria and fungi?
It’s the same thing. That’s probably what happened with the witch’s broom fungus that attacks cocoa trees. It may have come from the Amazon or from Africa. Some say it first appeared after a congress in Bahia that was attended by cocoa researchers and planters from all over the world. Someone brought it in – perhaps inadvertently, perhaps on purpose – and contaminated Bahia’s plantations.

Your PhD thesis was on the bacterium Xanthomonas campestris. Why did you choose that one?
After I graduated from ESALQ I was introduced to a now-deceased professor from the United States, Milislav Demerec, who knew bacterial genetics well and suggested I work with one that caused disease in plants. Xanthomonas campestris causes a disease in kale, cabbage, mustard plants, etc. I wanted to study Xanthomonas citri, which causes citrus canker. But I was advised not to do so because at the time – the 1960s – there were roadblocks and citrus canker occurred, for example, in the Botucatu region. When you came from Botucatu and neighboring towns, you were stopped at a roadblock. They used to open the trunk and if there were any oranges in it, you had to throw them out to avoid contaminating the Limeira area with citrus canker, which was a very important citrus region but that was yet to be affected by the disease. I decided to study X. campestris because if the disease turned up in Limeira they’d say I was the one who’d brought it in.

Was your objective to find an antibiotic for citrus canker?
It had to do with that. It just so happens that, if you treat the seed with streptomycin or kanamycin, you can reduce the incidence of the disease but resistant forms then appear. That’s the danger with using antibiotics in agriculture, just as in medicine. This was the period when many resistant organisms appeared. At Ceasa [an important supply center in the State of São Paulo], for example, they used to take carrots and put them in a drum full of streptomycin to avoid root rot. The problem was that they smelled of antibiotic, which people consumed along with the carrots. It was crazy. It was when I started thinking about this that I began working on resistance to antibiotics.

When you decided to do your PhD had you already decided on a scientific career?
Yes. Professor Friedrich Gustav Brieger, the head of department who influenced me, said that no one was working on microorganisms in Brazil. Brieger was German. He came here at a time when USP was bringing in academics to help set up the university and he was one of the pioneers in genetic studies here. In Germany, he had been a student of Carl Erich Correns, who rediscovered Mendel’s laws. Brieger said to me, “You need to study microorganism genetics, because there are people already studying the genetics of the fruit fly, human beings, plants and large animals…” Microorganisms generate in 20 to 30 minutes. Genetics study the transmission from father to offspring. Doing this in humans takes 20 years. With microorganisms, it takes 20 minutes. He also pointed me in the direction of bacterial genetics, but not in the health area. Brieger warned me, “You’re in an agronomy school, so study a plant pathogen.” Obviously, he was absolutely right.

Why did you choose to go to England to specialize?
I graduated in 1959. I finished my PhD in 1962 and, in 1964, I went to study abroad. Brieger suggested I go to England instead of the United States. In the States they were very good at bacterial genetics and I’d be part of a team in which each one did a little piece and I’d be dependent on them when I came back to Brazil. In England, on the other hand, I would work with fungi, something that in the United States they were not very good at, and I would have more independence. I went to the University of Sheffield and studied a fungus called Aspergillus nidulans. I was lucky to meet Guido Pontecorvo, an Italian professor. He had a great assistant, Professor Joseph Alan Roper. People from the British Council, which had given me a scholarship, suggested that I work with Roper, who was younger and more active in research, besides having been recently hired as the head of the Genetics Department in Sheffield. By then, Pontecorvo was only lecturing. The study model, Aspergillus, was a good choice. This fungus cannot be used in biotechnology, but it’s good for studying genetics because it doesn’t cause disease and is easy to grow. There are other Aspergillus: the A. niger can be used for making citric acid and it’s interesting in terms of biotechnology. A. parasiticus and A. flavus, on the other hand, produce toxins and are dangerous.

After this period in England, did you come back to ESALQ?
I came back in 1970 and the first theses I supervised at that time were all about A. nidulans. There was even a certain application in some cases because it has a cycle, called a parasexual cycle. This happens in fungi used in industry and can be used for producing antibiotics, citric acid and other things. As I was coming back to this area, for some time I helped Fermenta, which belonged to the Matarazzo family, in Santa Rosa de Viterbo [SP]. The Amália plant, which also belonged to the Matarazzos, produced citric acid – which has nothing to do with citrus fruit – and they called us in to resolve a problem. This acid was exported and is used to make candies; it’s also used in medication and soft drinks. I studied the situation along with my post-graduate students who ended up discovering that the parasexuality of the fungus was altering production. One of these post-graduate students managed to stabilize the fungi and solved the problem.

You have already said that, depending on the bacteria that act with Xylella fastidiosa, it might become weaker or more and more active. How was this discovered?
One of the hypotheses is that bacteria of the Methylobacterium and Curtobacterium type, which coexist with Xylella on the plant, each produce something that Xylella takes advantage of or that harms it. We discovered this after 2001, after the sequencing, which took place between 1997 and 2000. I was part of the Xylella Steering Committee, although I had not been one of the authors of the genome sequencing study. This committee used to receive projects so it could give its expert opinion. Sequencing was very good because it put Brazil on the map, internationally. But in addition to sequencing, we needed to do something to fight Xylella. That’s why the functional genome of the bacteria was unraveled in 2000. This was also funded by FAPESP, and in 2001 these two other bacteria that interact with Xylella were discovered.

EDUARDO CESARWhat makes them act differently?
The best hypothesis we have involves siderophores, the metabolites of microorganisms that capture iron and transfer it to other cells. Some cells need iron and removing it from the environment makes it difficult for Xylella to grow, which is the case with Curtobacterium. If, on the other hand, they release iron, like Methylobacterium, it gets better. But this is yet to be proved. Today, Wellington Luis Araujo’s group at the University of Mogi das Cruzes is studying this part.

So this hypothesis didn’t help solve the problem of the “amarelinho” disease, caused by Xylella?
No. I think that what it primarily solved was how to produce suitable insect-free saplings. It’s the insect that carries Xylella; it carries both good and bad bacteria. My opinion is that Xylella already existed in Brazil, but that something happened that caused an environmental imbalance. It might have been the weather, crop treatment or possibly the huge density of orange groves. This must have favored the growth of Xylella, which already existed in the United States, although there it affects grapes rather than oranges.

Are you one of those researchers who criticize yourself concerning the expectations you had for the so-called genomic revolution?
The tool is important. But we’re in the same position today as we were before – an effective vaccine against malaria and other diseases that both plants and people suffer from has not materialized. This is not the way we’re going to solve these diseases in the short to medium term.

But is this what you thought would happen at the height of the genomic boom?
No, it wasn’t. I thought it was all very much exaggerated. Molecular biology is more of a working tool than a solution. Of course, if I can, I’m not going to stop mapping out genomes, because there’s always information that helps. When the Xylella genome was sequenced, we saw that it had the genes of a gum similar to xanthan gum, which is the same as the ones found in Xanthomonas campestris, used in oil wells and for thickening foodstuffs, like ice cream and jelly. When they found these genes in Xylella I thought that the gum could be destroyed if, in an endophytic bacterium, we introduced a plasmid with an enzyme gene that would destroy the Xylella Igum. Xylella lives in the sap vessels inside the plant. It’s just as if we were to unblock these vessels by placing another bacterium in them that would destroy the gum. Isn’t that a great idea?

It seems to be…
Well, it didn’t help at all.

Because the main thing in this story is biofilm, which are sticky bacteria cells inside the vessels. Today, we know that the main issue is that the bacterium that produces the gum sticks to the plant vessels. It sticks hard there and doesn’t come out. For several years we’ve been trying this strategy and nothing’s happened. The plant’s fine. With all this going on it was discovered that the Xylella genome actually has this gene, but it’s not the main cause of the disease – the principle cause is the genes that make biofilm. As you can see, there are advantages to being familiar with the genome of bacteria. You can’t say that genomics has been useless.

What’s the other disease you studied, like citrus black spot?
This one’s not very serious. It’s more of a visual issue. When we buy oranges we don’t like them to have small black blotches. We always prefer attractive looking ones. In Europe they’re rejected right away if they have black spots.

But does it cause disease?
No. I mean, if they have a lot of it, it might, but it’s a disease that mainly prevents oranges from being exported. When we sent fruit to the Netherlands, for example, the ship would often arrive full and they used to refuse to accept it saying that it had the Guignardia citricarpa fungus that causes black spot. As this microorganism doesn’t exist in Europe they block its entry. We analyzed the case and saw that it didn’t have the fungus. It just so happens that normal fruit with some black blotches is one thing and the fruit that has the pathogen is something else. Visually they look the same. When the fruit arrived in Europe and they tested it they said it had G. citricarpa and sent it back because their test was inaccurate.

How did they do the test?
Under a microscope. We created a more accurate molecular test to differentiate the two situations. My group carried out the development, Fundecitrus paid for the patent and the kit was produced.

Was this kit sold?
When it was needed Fundecitrus did. This was the brainchild of Walter Maccheroni Jr., who is today with Canavialis, a company taken over by Monsanto. But he lost interest because Fundecitros saw that the objective was to bar the entry of Brazilian oranges in any way whatsoever in order to favor Spain, Europe’s major producer. Since black spot was no longer a reason for barring entry to Europe, they found a fungicide that was used in Brazil and prohibited there. What they wanted was to prevent the entry of oranges and they managed to achieve this.

You’ve worked in many places, but are you still involved with ESALQ?
I’ve been asked to work in various places. In 1974, for example, I went to Campinas State University when Zeferino Vaz invited Professor Brieger to put together a genetics group, and he took professors from ESALQ. I was seconded to them for four years. In 1978 I returned to Piracicaba and in 1980 I was seconded to the University of Brasília to put together their genetics center. A little earlier, between 1979 and 1980, I had done a post-doctoral program in Nottingham, England, and then another in Manchester, from 1987 to 1989, on molecular biology, because I was not strong in that area. In 1990 I returned and became a dean at ESALQ, from 1991 to 1994.

When did you retire?
In 1995. Since then I’ve been freer because I’ve not had to worry about administration. So I’ve been doing more research. This was the period when I worked with Xylella.

But you’ve remained an adviser for post-graduate degrees, haven’t you? You’ve been responsible for 98 Masters and 68 PhDs…
The only way of doing good work is to have a specialist work force and this work force is graduate students. The secret is to always get the best ones. I’ve been advisor to people from Piracicaba, Campinas, Brasília, Mogi das Cruzes and Caxias do Sul, where I used to spend five days a month back in 1996. I’ve even been a tutor to students from Recife, where I’ve never had any links with the university. I used to give post-graduate lectures at least once a year there and students always used to come and work with me in Piracicaba and would then return to Recife. These numbers are nothing to be amazed about. You only have to work it out: I’ve been a graduate studies advisor since the early 60’s. That’s almost 50 years. The average is more or less three a year. That’s not that many…

Do you still have students?
I have 12. Most of them are from Manaus, which is short of advisors in various areas.

Why did you go and work in Manaus?
In 2002 they set up the CBA Biotechnology Center of the Amazon, under the auspices of the Brazilian Molecular Ecology Program for the Sustainable Use of the Biodiversity of the Amazon, which is linked to the federal government. But there have been a lot of problems since then and the place lost direction somewhat, until they started calling in researchers, generally retired ones, because they had no means of hiring people and these researchers had to take others with them, mostly people at the beginning of their careers. They invited people from USP, from the Butantan Institute, from the Federal University of São Paulo… I went in 2005.

To which ministry is the Center attached?
There are several ministries, including those of Industry Development and Foreign Trade, Science and Technology and the Environment. But it’s Suframa [Development Superintendence of the Manaus Free Zone] that contributes the most, financially. There are still very serious management problems with the federal government. I mean, we do our part as far as we’re able. There are eight coordination offices. I only look after microbiology.

Are all the students from Manaus?
From there and from other states. But no one has been hired. They’re all scholarship holders. The young people go there full of enthusiasm, but after one or two years they get full-time jobs and leave.

How do you see the Amazon in this microbiology area?
We’re finding fungi and bacteria on many plants and they’re kept in a microbial germplasm bank. We bioprospect what can be collected. What’s a good nitrogen fixer” What’s good for dissolving phosphate” Is there something good for killing insects or for putting an end to plant disease” Is there something that produces antibiotics or antitumor drugs” That’s more or less what we do.

And have they already discovered something promising?
There are substances that seem to act well against the tuberculosis bacillus, for example. We collected some plants they say serve as medicine for various illnesses, including tuberculosis, and we analyzed them and saw that there really are some microorganisms in the plant that attack mycobacteria.

Is there any possibility that these studies will result in some innovation?
The objective of the CBA is to provide services and technological innovation; discovering substances with good potential and handing them over to a company to develop. Pure research is already being done at Inpa [National Research Institute of the Amazon], which has a tradition in research going back many years. Human resources training is done by the Federal University of Amazonas, which has graduate courses.

Do you still have your laboratory at ESALQ?
I still work there, although I’m retired. They named the laboratory after me in tribute. But it’s annoying because those who don’t know me think I’m already dead.