Lethal symbiosis

Biological insecticide uses a partnership between bacteria and nematodes to fight sugarcane pest

Miguel BoyayanNematodes ready to attack the sugarcane weevilMiguel Boyayan

A biological insecticide capable of fighting the sugarcane weevil (Sphenophorus levis), a pest that can destroy up to 30 tons of plants per hectare and causes major losses for the sugarcane crop, is ready to be produced industrially. The bio-insecticide that may become an ally in  increasing  fuel alcohol productivity was developed in the Biological Institute, Campinas unit, in São Paulo State, and is based on minute worms, called nematodes that, when sprayed onto the soil, seek out the weevil. This hunt occurs because nematodes have chemical receptors in the cephalic region that are capable of locating a host insect by detecting excretion products liberated into the environment and by perceiving temperature variations and levels of carbon dioxide gas that occur when the two organisms get close to each other. As soon as the nematodes find the target they penetrate the insects’ apertures and liberate a bacteria they carry in their intestine that is responsible for causing septicemia and the death of the weevil in 48 hours.

After the weevil dies the nematodes start feeding on the bacteria themselves and reproduce within the insect. This scenario will last for up to three generations of nematode within the insect, after which they begin to suffer from lack food and the nutrients necessary to continue their life cycle. At this point the worms suffer a stimulus and are converted into infectious juveniles, a phase in which they store the bacteria in their intestines and are ready to seek out new weevils. Nematodes are called entomopathogenic, because they have a symbiotic relationship with the bacteria that are pathogenic for the insects. In this association the nematodes act like a vector of the bacteria, which in turn provides food for the minute worm.

Employed in more than ten countries, the biological control technique using nematodes was adapted to  Brazilian conditions by a group of researchers from the Biological Institute, which is linked to the Department of Agriculture and Supplies of the State of São Paulo. “Depending on the species,  the nematodes vary in behavior. There are groups that remain more on the surface of the ground and act against insects that are more mobile and others that attack insects that live underground, and that are not very mobile”, says agronomist and entomologist, Luis Garrigós Leite, from the Central Experimental Center of the Biological Institute, who started the research with nematodes and is responsible for the project.

The research that began in 2000 in the Biological Control Laboratory was given FAPESP support in 2003, as part of the Technological Innovation Program in Small Companies (Pipe) program. The partner company is Bio Controle, from São Paulo, which operates in the pest monitoring and control products market. Initially, the company’s partners wanted to import industrially-produced nematodes from England, an alternative that proved to be unfeasible because the sale price here in Brazil would have been close to US$ 200.00 a hectare. Furthermore, there was no guarantee that the imported product would be efficient in combating Brazilian pests. When the partners of Bio Controle found out that research in this area was under way, they contacted the researchers responsible.

Soil pests
The result was that they formed a partnership to carry out the project. “In the first phase we showed that it was possible to produce the nematodes on an industrial scale and then we began working on production and formulation”, says Leite. After selecting the nematodes and evaluating the potential market for the product the choice was for soil pests that attack sugar-cane. “Due to expansion of the crop and machine-harvesting as a result of the ban on burning, there are today more than five soil pests that cause damage in sugarcane crops”, says Leite. With mechanized harvesting the machine cuts the cane in the field and chops the straw, which is left on the ground, forming a thick covering. Too much straw results in a humid and protected environment that is ideal for pests like the sugarcane weevil to proliferate in. The insect, which is currently found in more than 40 towns around Piracicaba, in the State of São Paulo, could be found in just 14 towns in the same region back in 1989. Fighting the problem with chemical insecticides has not prevented an increase in pest populations. “Spending on chemical products is at least R$180.00 per hectare”, says Leite. Initially the bio-insecticide should cost around R$70.00 to treat one hectare, although the real price is estimated at round R$100.00.

Biological InstituteProducing nematodes of the genera Heterorhabditis and Steinernema on sponges and ready for use in the fieldBiological Institute

The process for obtaining the nematodes on a large scale starts by multiplying the bacteria on which they feed in a suitable environment based on cow’s liver. Then it is necessary to synchronize the production of bacteria with that of the nematodes, an important stage in the process. Nematodes in a suitable concentration are introduced to the bacteria that are growing in soaked polyurethane sponges, a porous material that offers biological support and ideal oxygenation for the organisms to reproduce. The subsequent steps of the process are collection and formulation, which must be capable of maintaining the nematodes in their juvenile stage – an almost hibernation-like state – until they are sprayed onto the plantations. The formulation is in the form of a powder or gel to be diluted in water.

Aggressive nematode
The Sugar-cane Plantation Technology Center (CTC) in Piracicaba helped select the nematodes to control the weevils. “We found a pretty aggressive nematode for these weevils, which attacks not only the larva, but also the adult”, says Leite. In relatively low nematode doses per hectare compared with most of the doses recommended in the United States and Europe, more than 70% of the insect larvae, which eat the plant’s rhizome in particular, and at least 25% of the adults were killed.

Selection started with the collection of nematode-infested soils that were taken to the laboratory. Today the project has a nematode bank with more than 30 separate species of the genera Steinernema and Heterorhabditis. “A species belonging to the Steinernema genus was selected and assessed in three field trials carried out at the São João Refinery, in Araras, and the Costa Pinto refinery, in Piracicaba; it produced similar results to the chemical insecticide currently used for fighting the sugarcane weevil”, says Leite. In the tests it was proven that in those areas where the nematodes were sprayed, cane production 17 tons greater than in untreated areas that were therefore susceptible to attack by the insects.

The researchers believe that the application of nematodes in the field should, over time, stabilize the insect population. “They survive well in the soil from one year to the next”, says biologist Carmen Maria Ambrós Ginarte, coordinator of the PIPE Project. In order to assess the nematode’s endurance and effect over a four year period with annual applications after   planting, other trials are being carried out. “We want to compare the biological control results with those of chemical control to find out the increase in production and how much damage to sugarcane plantations has been  avoided”, says Carmen Maria.

The Dedini Group, which produces  sugar, alcohol and equipment for the sugar and alcohol industry, whose headquarters are in Piracicaba, is a partner in this initiative. “They want to have an assessment of areas treated with nematodes on a semi-commercial scale vs. those that are untreated”, says Leite. The trials were conducted on 50 hectares and are now being extended to a further 50 hectares in plantations belonging to the Iracema Refinery in Iracemápolis. In addition, tests to assess the effectiveness of nematodes on other pests are being carried out. One of them is studying the action of Heterorhabditis against the larvae of Bradysia, a 2 mm long fly, considered to be a pest in ornamental plant nurseries. The adult form causes no problems, but when the larvae hatch from the eggs laid by the flies they attack the root system of plants, damaging them in their growth phase. In laboratory tests a selected species of this nematode caused a more than 70% mortality rate among the larvae and pupae of this insect.

The Project
Assessment of methodologies and techniques for the industrial production of entomopathogenic nematodes and market research for the marketing of these agents (nº 03/02137-1); Modality Technological Innovation Program in Small Companies (PIPE); Coordinator Carmen Maria Ambrós Ginarte – Bio Controle; Investment R$ 332,188.65 (FAPESP)