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Natural insect bank

Collection of fungi, viruses and bacteria help combat agricultural pests

A collection of test tubes containing samples of fungi, viruses, bacteria, nematodes and protozoa is changing the way pests that destroy Brazilian agriculture is combated. Arriving in a constant stream from all over Brazil, these samples comprise the inventory of the Microorganism Bank that was set up at the Biological Control Laboratory of the Experimental Center of the Biological Institute in Campinas. Based on these samples, natural biological insecticides can be bred which will substitute chemical pesticides in the fight against nation-wide pests such as the migratory locusts, the insect  “percevejo-de-renda”(Leptopharsa heveae)  which attacks rubber plantations, the beetle “moleque-da-bananeira” (Cosmopolite sordidus) which attacks the banana, and the citrus borer. And the advantage is that they are non-toxic. “There is no anecdotal evidence of plants, men and animals having been attacked by entopathogenic micro-organisms”, says José Eduardo Marcondes de Almeida, the curator of the bank. There is one more reason for celebration: “The bio-insecticides can be administered in the same way as chemical pesticides, but without any specialized equipment for the purpose.”

The entopathogenic microorganisms – which do not affect plants or animals but cause disease in insects – are kept in cold storage which resemble vaccine production centers, such is the level of sterilization. These micro-organisms form the basis for the raw material which is supplied for the studies in progress for developing biological pest control, taking place in São Paulo, Paraná, Bahia and Pernambuco (see box). It is also from this material that the  Oldemar Cardim Abreu Entopathogenic Micro-organism Bank – so-called in honor of the research scientist at the Experimental Center in Campinas who, at the end of the seventies, began studies into the production and storage of the Metarhizium anisopliae fungus – produces six natural pesticides. And this is not all: last year, the bank produced 1.5 tons of  natural biological pesticides, sold to farmers and agribusiness throughout the country, both in liquid and solid form at a price close to cost (R$ 20 per liter or R$ 15 per kilo).

According to Antonio Batista Filho, who coordinates the bank and directs the Experimental Center, these results are due to major turnabout in the history of the bank. Created in 1982 by an agronomist, Benedicto Pedro Bastos Cruz, its progress can be described as discreet until August last year. This was when, with R$ 44.3 thousand in financial support from FAPESP, the bank began to be reorganized as part of the Formation of a Entopathogenic Micro-organisms Bank in the Biological Control Laboratory under the coordination of the Experimental Center of the Biological Institute. The bank was given a major boost with new insect breeding rooms, incubators, microscopes and refrigerators for storing micro-organisms, such that the numbers in the collection rose from 63 to 260 isolated samples.

Each type of micro-organism gets the treatment it deserves. Fungi samples are stored in test tubes in a culture with sterile mineral oil in a refrigerator or in eppendorfs (small plastic tubes with lids) in the freezer. With the major changes introduced last year, the 63 samples – previously conserved by an old  technique in pre-cooked rice – were re-isolated and  are now stored in the eppendorfs and culture tubes.

The bacteria are also held in culture tubes in the refrigerator, the viruses held in suspension and dead insects in the freezer. Meanwhile, the nematodes are preserved in cultures and the protozoa, in dead insects in the freezer. As the microorganisms may be needed for new research at any moment, they are maintained alive, albeit in a state of dormancy – almost hibernation.

Almeida was 14 and still lived with his parents in Campinas when he decided: from then on, he would seriously study the mechanisms that control agricultural pests, without polluting the environment and contaminating plants, animals and man himself. In other words, an alternative to chemical pesticides that were beginning to be looked upon unfavorably. With this background, it is easy to understand the ill-concealed pride with which today, at 32, he displays the inventory of samples in the Micro-organisms Bank.

Almeida fulfilled his childhood dream, but as an agronomist, he has still not reached his objective. He says that for the bank to support the demand from all the programs for the biological control of pests, it will be necessary to obtain at least 500 fungi isolations, 25 for viruses, bacteria and for nematodes of the most important species. That is almost double the existing inventory. However, this is not an impossible mission: every week, an average of ten new microorganism samples is received.

Up to now, at least 80% of the micro-organisms in storage are in the form of fungi. These dominate because they act on insects by ingestion and contact. For this reason, they are easier to handle during collection, identification, isolation and production of the natural pesticides. But it is not always so easy.  The development of isolation techniques, conservation, handling of viruses, bacteria, nematodes and protozoa, that provoke infections when ingested by insects, are still at a preliminary stage of development. “In a dead insect”, Almeida reminds us, “bacteria deteriorate in two or three days, while the deterioration of the viruses takes up to ten days”.

The team of five research scientists and two technicians traveled throughout the State of São Paulo in search of samples to establish the inventory. They collected the material from different regions because on many occasions, microorganisms of the same species have genetic peculiarities and attack different types of pest. As he speaks of the origins of the inventory, Almeida does not fail to emphasize the exchange of samples among similar banks of the College of Agriculture Luiz de Queiroz (Esalq), of the University of São Paulo (USP) and the São Paulo State University (Unesp) in Jaboticabal.

The Experimental Center supplies sample material to banks which are being set up – such as the State University of Londrina, in the state of Paraná, and the Federal University of Lavras, in the state of Minas Gerais. It also maintains interchange agreements with overseas centers such as the University of Utah, in the USA, the Ministry of Agriculture and Fisheries in Costa Rica, and the Cuban Institute of Vegetable Sanitation.

Only those public institutions that are researching microbial control of insects or the genetic improvement of micro-organisms, which are beneficial to agriculture, receive sample material. There is one indispensable precaution: a documented signed by researcher responsible for the institution that the material will only be used for research. Supply to companies that actually produce the natural pesticides occurs only after the signing of an agreement for royalty payments.

Current research is focused on five species. Three are fungi: Metarhizium anisopliae, Beauveria bassiana and Sporothrix insectorum. Additionally, there is a virus, Baculovirus. The fifth is a bacterium, Bacilus thuringiensis. Each one of them merits a quick description. Metarhizium attacks the largest number of insects and pests. Two examples: the citrus borer, which does significant damage to orange and lemon trees, and the migratory locust, which causes grass for animal forage to turn yellow, thus adversely affecting meat and milk production.

Beauveria is another fungus with wide-ranging action: it infects the coffee and banana borer pest, among others. The Sporothrix controls the “percevejo-de-renda” (leptopharsa heveae) which is the main pest to attach rubber plantations in the State of São Paulo. The Baculovirus is the virus which is central to one of the world’s leading programs for biological control for combating the soybean lizard – a pest which eats the plant leaf and drastically reduces output. Finally, the Bacilus thuringiensis controls various types of lizard, among which, the soybean lizard and the tomato moth.

Banana Plants
The discovery of more virulent micro-organisms has enabled the formulation and production of specific natural pesticides. This is the case with the CB 66, a strain from the fungus, Beauveria bassiana, that attacks the red mites of the Paraguayan tea plant. It also controls the borer, known locally as “moleque-da-bananeira” (cosmopolite sordidus), that perforates the root and the trunk of the banana plant. The “moleque-da-banana” further transmits such diseases as the one caused by the Fusarium oxysporum f. sp. cubense which also attacks the banana plant and contaminates the soil and is a growing danger.

“In São Paulo, the varieties of apple (maça) and silver (prata) banana are nearing extinction due to the Fusarium oxysporum f. sp. cubense”, informs Batista Filho. Since 1995, the Biological Center has played a part in the use of the Beauveria fungus in the combating of the banana borer. The fungus was originally developed for the banana growing areas of the Ribeira Valley in São Paulo. The use of this natural pesticide has contributed to preserve the ecosystem and assure the quality of life for the inhabitants of the region.

The Ribeira Valley experience was a testimony to the Bank’s significance and showed the way to new needs. Since last year, the research scientists have been working on the intensive production of bioinsecticides based on live fungi with funding of R$ 50 thousand from FAPESP for the project, “Development of Production Processes and Formulation of Beauveria bassiana, Metarhizium anisopliae and Sporothrix insectorum”. The objective is to solve a problem: the problem of conservation which will allow these products to be kept at room temperature where no scheme for refrigeration is available – a not uncommon situation in rural areas in Brazil. With this in mind, already in 1997, the team from the Biological Center formulated a natural oil-based pesticide with Baculovírus anticarsia, which showed an efficiency that was over 80% for the control of the soybean lizard. And the conservation goal was reached: the product can now be stored in ordinary cupboards for up to two years.

High Production
Once it is certain that a formulation is going to work, production grows rapidly. An example: during the last harvest, the rubber latex producers of the state of São Paulo, the largest domestic producer, sprayed 5 thousand hectares of rubber plantations with 5 thousand liters of liquid natural pesticide, CB 79. Prepared with a strain of the fungus Sporothrix insectorum, CB 79 controls one of the most undesirable pests to attack rubber plantations, known locally as “percevejo-de-renda” (leptopharsa heveae) which reduces the plants’ levels of photosynthesis, adversely affecting rubber latex output. In addition, more than 460 kilos of the E9 natural pesticide made from the Metarhizium anisopliae fungus has been used to combat the citrus borer and the migratory locust, mainly in the Paraíba Valley.

Natural pesticides for the biological control of pests are also produced by companies, in some cases using technologies transferred by research institutes such as the Biological Center. The sales amount to significant values. Almeida estimates that 20 tons of natural pesticide with Baculovírus is applied on more than a million hectares of soybean plantations and worth more than US$ 2 million annually. The use in Brazil of mixtures containing Bacillus thuringiensis – one of the most important bacterium for controlling several types of lizard and believed to account for 60% of world sales of natural insecticides  –  is just shy of 160 tons annually and produce revenues of US$ 2.5 million. Every year, agriculture in Brazil consumes something around 66 tons of fungus-based natural insecticides: 55 tons of Metarhizium anisopliae, 3 of Beauveria bassiana and 8 of Sporothrix insectorum, equivalent to sales of approximately US$ 1 million.

Genetic Improvement
As soon as possible, the team at the Biological Center plans will begin studies for the genetic characterization and improvement of the micro-organisms. The other objective: to take steps to establish the patents to protect the natural insecticides. Because the evidence is already clear: there is still huge potential to be exploited. According to Almeida, there are more than 1 million species of identified insects in the world that are known to cause at least one kind of disease.

“In the twenty-first century, methods of biological control will be one of the main methods of fighting pests”, says the scientist who, while excited by the prospect, remains firmly focused on the reality of the situation. According to him, the environmental impact must be examined in the light of the number of micro-organisms and the area where they are applied. “For starters”, he says, “we believe that the environmental impact is very limited or practically zero when compared with the use of chemical pesticides.”

• Antônio Batista Filho, born in the city of São Paulo, 42, graduated as an agronomist in 1981 and concluded his Ph.D. 1997, in Entomology at the College of Agriculture Luiz de Queiroz (Esalq) of the University of São Paulo (USP). He is coordinator of the Oldemar Cardim Abreu Entopathogenic Micro-organisms Bank and director of the Experimental Center of the Biological Institute of the Department for Agriculture and Supply of the state of São Paulo in Campinas, where he is a research scientist.

• José Eduardo Marcondes de Almeida, born in Campinas, 32. Graduated as an agronomist in 1990 at the Federal University of Lavras, State of Minas Gerais, and concluded his Ph.D. in Entomology two years ago at College of Agriculture Luiz de Queiroz (Esalq) of the University of São Paulo (USP). He is curator of the Micro-organisms Bank and has been doing research work at the Biological Institute since 1998.

Project: Formation of a Bank of Entopathogenic Microorganisms in the Biological Control Laboratory at the Experimental Center of the Biological Institute.
Investment: R$ 44.327,50

Good news from all sides
There are eight projects in progress based on micro-organism samples sent by the Biological Institute of Campinas. In one of the laboratories of the Institute itself in Pindamonhangaba, research scientist and agronomist, Hélio Minoru Takada is testing the control of the rice water weevil, Oryzophagus oryzae, with the Beauveria bassiana CB 74 and has proved that it causes a 70% mortality rate in the population of the pest.

At the State University of Campinas (Unicamp), another research scientist at the Biological Center, Leila Barci, a biomedical specialist, selects micro-organisms from two fungi, the Beauveria bassiana and the Metarhizium anisopliae, for the control of cattle ticks. This is the theme of her Ph.D. thesis.

At the University of West Paraná, in Cascavel, the biologist Renato Cassol, under the guidance of Professor Luiz Francisco Alves, studies the control of the striped mite which attacks the mate tea plant. As a rule, chemical pesticides cannot be used in the cultivation of this plant since the use of such would contaminate the leaves which are consumed directly.

Almeida himself is coordinating a project for the control the cicada which attacks the roots of sugar cane causing the yellowing of the crop in the state of São Paulo and losses of up to 30% in tons per hectare of sugar cane. The agronomist, Luís Garrigós Leite, of the Experimental Center of the Biological Institute, is preparing his Ph.D. at the University of Utah into the development of methods to produce three species of fungi pertaining to the Entomophthorales group. He intends to use these in the combat of cicadas and mites. Leite has already discovered the effect of salts, vitamins, amino acids and sugars in the growth of these species.