Patent Description:
In the current world-wide agricultural scenario, the production profits have been many times associated to the gradual productivity increases without a corresponding increase in the cultivable area. Such productivity increases have been attained by means of significant advances in the cultivation techniques, use of varieties which are more suited to biotic and abiotic factors, adequacy of the nutritional need of the plant and, nevertheless, the mitigation of damages caused by agricultural pests.

Amongst such advances, the pest control is still considered as the biggest challenge in the maintenance of crop productivity, wherein several techniques can be employed, with a higher or lower degree of efficiency, but generally the use of chemical defensives is the most used method.

However, for the control of phytonematodes, the use of agrochemicals has frequently shown unsatisfactory results, and additionally the excessive use of chemical nematicides has frequently caused intoxication of humans and/or animals, concomitantly with the contamination of the environment. Such events have led to an increase of the public opinion on the use of nematicides, and consequently, to a never-ending search for safer handling techniques.

Thus, alternative controls for phytonematodes have been employed whenever possible, but adversities inherent to the biology of said organisms usually make the use of some techniques unfeasible. For example, varieties of resistant cultivars are hardly available, while the rotation culture is impracticable by virtue of the costs or the wide range of hosts for some species (Bird et al.

In this context, the biological control of phytonematodes by using microorganisms is considered as a viable option. Its main advantage, compared to other technologies, is again the possibility of exploring different ways of action of agrochemicals (Zucchi et al. Indeed, mechanisms of action such as antagonism and parasitism have been widely used in commercial products.

Antagonism is usually the predominant action of bacteria. In addition to the direct effect on the mortality of phytonematodes, such nematicidal compounds can act directly on the emergence of eggs or mobility of nematodes, and also cause indirect effects such as changes in root exudates or induction of resistance (Sikora & Hoffmann-Hergarten, <NUM>;Hasky-Günther et al.

The use of bacteria as biological control agents is more promising in the case of endoparasites phytonematodes (Hallmann et al. <NUM>) such as, for example, Meloidogyne graminicola. Bacillus megaterium reduced <NUM>% of the penetration and formation in boughs of such phytonematodes in rice roots, besides diminishing <NUM>% of its migration to rhizosphere and reducing <NUM>% the emergence of eggs (Sikora & Padgham <NUM>). Higaki & Araujo (<NUM>), the use of Bacillus subtilis for the control of Pratylenchus brachyurus was as good as the chemical treatment using Abamectin, which result was extremely small populations of such phytonematodes in the soil, about <NUM> phytonematode/cm soil, reducing about <NUM>% of this population in view of the standard treatment.

Other results in this line of research, also using Bacillus subtilis for treating seeds, is evidenced by Higaki & Araujo (<NUM>), showing that this alternative handling technique caused reductions in the order of <NUM>% of the population of Pratylenchus spp. <NUM> days after sowing, adding <NUM>% in productivity.

Similar results had been attained by Higaki (<NUM>) using Bacillus subtilis for controlling Rotylenchulus reniformis and Pratylenchus brachyurus in cotton plants, said treatment using microorganism resulting in reductions higher than <NUM>% in the population of said phytonematodes in the roots of the crop.

Other interesting data reported by the author is that the plants treated with said microorganism exhibited increments in the fresh mass of roots and in the aerial portion in the order of <NUM> and <NUM>%, respectively, in view of the standard treatment. In accordance with Araújo et al. (<NUM>), the mechanisms of action responsible for the promotion of plant growth can be linked initially to the direct inhibition of the pathogen and induction of systemic resistance, amongst others. It is usually difficult to recognize the mechanisms and associate same with the direct growth promotion, since more than one mechanism is produced by the bacteria.

Despite these advantages, most of the existing products are based on the premise of exploring a single microorganism to control phytonematodes. As disclosed, some biological control agents have more than one mechanism of action which can act directly or indirectly on the target phytonematode. However, associations among several biological control agents, thus extending the spectrum of action of such microorganisms against phytonematodes, have not been widely explored yet.

In a research conducted in specialized data bases, documents related to the composition for the biological control of phytonematodes have been found. One of said documents, <CIT>, is related to a composition comprising Bacillus subtilis (DSM <NUM>) and Bacillus licheniformis (DSM <NUM>) having a nematicidal effect against phytonematodes in plants and/or the habitat thereof, the use thereof, and a process for the preparation thereof, the use of Bacillus subtilis (DSM <NUM>) and Bacillus licheniformis (DSM <NUM>), and processes to control, fight and provide specific resistance to phytonematodes, and a kit. Also <CIT> and <CIT> disclose methods of controlling phytopathogenic nematodes with a combination comprising Bacillus subtilis and Bacillus licheniformis.

This approach seems to be more efficient, since it increases synergically the individual effect of each isolate. The disadvantage is that it is based only on antibiose for the control of the target pest, what may occasionally lead to the selection of resistant individuals. Despite this, these examples illustrate a possible trend in the biological control of phytonematodes, that is, the use of complex biological compositions to mitigate the deleterious effect of said organisms.

The present invention refers to a composition consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, concomitantly with additives and excipients, as defined in the appended claims. The compositions have nematicidal properties for the control of phytonematodes in plants. Thus, the object of the present invention is to develop compositions having more than one mechanism of action which are effective against phytonematodes. Said compositions involve the use of different species of bacteria (antibiose, egg parasitism and reduced phytonematode ability of orientation).

With the composition for biological control of phytonematodes thus obtained, the following advantages can be attained:.

The present patent application is related to a "COMPOSITION FOR BIOLOGICAL CONTROL OF PHYTONEMATODES", more precisely a composition of bacteria with nematicidal effects in damage mitigation and control of phytonematodes on cultivable plants.

According to present invention, the composition for biological control of phytonematodes comprises the following microorganisms at a concentration in colony forming units per gram (c.

The isolates were identified and classified by the Coleção Brasileira de Microorganisms de Ambiente e Indústria (CBMAI/UNICAMP), where they have been deposited.

The composition has the following concentrations:.

The composition according to the invention further comprises additives selected from the group consisting of dispersants chosen from the group consisting of water-soluble ionic polymers, water-soluble anionic polymers, surfactants selected from the group consisting of anionic surfactants and non-ionic surfactants, and the combinations thereof.

The composition according to the invention further comprises excipients selected from the group that consists of: silicas, talc, bentonite, carbohydrates, carbonates, casein, milk serum and powder milk, and the combinations thereof.

The composition should be used as a formulation in a wettable powder. However, other formulations containing said microorganisms such as emulsions, concentrated suspensions, and granules also can be used.

A composition containing <NUM>% Bacillus subtilis, <NUM>% Bacillus licheniformis, <NUM>% Bacillus amyloliquefaciens, <NUM>% acrylic styrene polymer, <NUM>% anionic surfactant, and <NUM>% inert component was formulated to evaluate its efficiency in the control of phytonematodes. The examples below illustrate the use of this composition:.

To evaluate the effect under field conditions of the biological formulation containing isolates of Bacillus in the control of Meloidogyne incognita, applied to plantation furrows.

Experimental layout used: casualized blocks with six treatments and six repetitions (<NUM> portions). The experimental portions were <NUM> wide (eight cultivation lines) and <NUM> long, totaling an <NUM><NUM> area.

Treatments and form of application: The treatments on plantation furrows before the sowing were applied only once. For the application, a CO<NUM> pressurized costal spray was used, with a constant pressure of <NUM> PSI, connected to a common lance having a fan type spraying nozzle, and a broth volume equivalent to <NUM>.

Sampling and evaluation of the effectiveness of the formulation: Soil and root samples were collected randomly, in five different points of the useful portion making out a sample comprising approximately <NUM> soil and <NUM> of root. Said collections were carried out at a depth of <NUM>-<NUM>. For the quantification of eggs and younglets of M. incognita, the samples of each experimental portion were sent to the nematology laboratory. The extraction of the target in the soil was carried out through the centrifugal flotation technique in a sucrose solution proposed by Jenkins (<NUM>) in <NUM><NUM> of the soil, wherein the evaluations were made previously <NUM> and <NUM> days after sowing. For the quantification of nematodes in the roots, the liquefier technique proposed by Coolen & D'herde (<NUM>) was used in <NUM> of root, said evaluations being made <NUM> and <NUM> days after sowing. The reproduction factor, which is the relationship between the final population (Fp) and the initial population (Ip) of phytonematodes, was calculated <NUM> to <NUM> after sowing, in accordance with the formula proposed by OOSTENBRINK (<NUM>), where FR=Fp/Ip. When the plants bloomed, the height of <NUM> plants in the center of each experimental portion was evaluated, by measuring the length of the stem from the soil level to the last leaf of the plant with the aid of a measuring device. To estimate the productivity of the crop in kilograms per hectare, the grains were harvested and weighed in <NUM><NUM> and the humidity was set to <NUM>%, on <NUM>/<NUM>/<NUM>. When required, the raw data of the evaluations were changed or had their outliers disregarded in order to attain the normality of the data and then they were submitted to the variance analysis. The comparisons between the averages were made through the test Scott-Knott test (p<<NUM>) (<NUM>) and the effectiveness of the treatments were calculated according to Abbott (<NUM>).

In the previous monitoring and along the evaluations, the presence of natural enemies has not been detected in significant amounts and frequency in the plants of the experimental area, thus making it impossible to estimate the effect of the treatments in a study on the dynamics of this population. Further, it was verified that there was no phytotoxicity caused by the application of the treatments in the study that would compromise the growth and development of soy plants (Table <NUM>).

In the plant height evaluation (Table <NUM>), the applied treatments are not statistically differed from the standard, exhibiting height of plants that varied from <NUM> to <NUM>. Rhizobacteria can promote the growth of plants (LUZ, <NUM>), however, in areas highly infested by nematodes, the growth of the plants can be jeopardized and said effect may not be observed.

In accordance with Table <NUM>, in connection with the effect of the applied treatments on the number of young and/or adult plants in <NUM><NUM> of soil, on the <NUM>th day after sowing a significant difference between the treatments could be noticed. Doses of <NUM>, <NUM>, <NUM>, <NUM>. ha-<NUM> of the biological formulation exhibited a higher control compared to other treatments, with an efficiency of up to <NUM>%. At the <NUM>th day after sowing, a higher differentiation between the treatments was noticed, with doses of <NUM> and <NUM>. ha-<NUM> of the biological formulation higher than the standard and doses of <NUM>, <NUM> and <NUM>. ha-<NUM> statistically higher than all the other treatments and high efficiency ranging from <NUM>% to <NUM>%. These results indicate a long-lasting effect of the biological formulation in the ground, making possible to better control the pest and, consequently, more protection throughout the cycle of the crop.

Table <NUM> presents the effect of the applied treatments on the number of young and/or adult plants in <NUM> of root. It was evidenced that to there was a clear distinction between the treatments on the <NUM>th day after sowing, where it was possible to separate them into two groups: a) treatments at doses of <NUM> and <NUM>. ha-<NUM> that despite being statistically higher than the standard showed a low efficiency, and b) the group with the treatments at doses of <NUM>, <NUM> and <NUM>. ha-<NUM> of the biological formulation that showed a higher control efficiency, attaining a reduction in the population of nematodes of up to <NUM>% in the roots.

In the evaluation conducted at the <NUM>th day after sowing, the treatment at a dose of <NUM>. ha-<NUM> of the biological formulation showed the highest control efficiency (<NUM>%) which is significantly superior to the other treatments. Next, doses of <NUM> and <NUM>. ha-<NUM> also showed a high control efficiency (<NUM>% and <NUM>%, respectively), and the corresponding treatments at doses of <NUM> and <NUM>. ha-<NUM> showed the lowest efficiencies, even though they were statistically superior to the standard.

The results of the control of nematodes in the root are similar to the results attained in the control of nematodes in the soil (Table <NUM>), where the highest control efficiencies were observed at the <NUM>th day after sowing, indicating the long-term continuous effect of the biological formulation.

The consequences of the control of the nematodes in boughs on the reproduction factor are presented in Table <NUM>, where it is seen that at the <NUM>th day after sowing the standard treatments and biological formulation at a dose of <NUM>. ha-<NUM> were not statistically different from one another. However, doses of <NUM>, <NUM>, <NUM> and <NUM>. ha-<NUM> showed a significantly higher efficiency, with a reduction in the reproduction factor that varied from <NUM>% to <NUM>%.

At the <NUM>th day after sowing, the standard treatment and biological formulation at the dose of <NUM> and <NUM>. ha-<NUM> did not show a significant difference between one another, but only the treatments at doses <NUM>, <NUM> and <NUM>. ha-<NUM> were significantly superior to the standard, showing a control efficiency between <NUM>% and <NUM>%.

As to the productivity of the crop, there was no statistical difference between the applied treatments and the standard, but treatments containing the biological formulation at a dose of <NUM>. ha-<NUM> provided a <NUM>% increment in the productivity of the crop (Table <NUM>).

In accordance with the results attained, the biological formulation exhibits nematode efficiency control in boughs (Meloidogyne incognita) in soy plants, and can be used in the control of this pest.

None of the evaluated treatments caused phytotoxicity to soy plants (Glycine max), cultivar NA <NUM> RG. The treatments with the biological formulation at doses of <NUM>, <NUM>, <NUM> and <NUM>. ha-<NUM> exhibited a control of nematodes in boughs (Meloidogyne incognita), with a reduction of <NUM> to <NUM>% and of <NUM>% to <NUM>% in the number of young and/or adult plants in the soil and the root, respectively. Further, it reduced the reproduction factor by <NUM> to <NUM>%, and can be considered as an efficient tool in the pest control of soy plants.

To evaluate the effect under field conditions of the biological formulation containing bactericides for controlling Pratylenchus brachyurus applied by way of treating the seeds.

Experimental layout: the experimental layout use was: casualized blocks, with six treatments and six repetitions (<NUM> portions). The experimental portions were <NUM> wide (eight cultivation lines) and <NUM> long, totaling a <NUM><NUM> area. When required, the data of the evaluations were changed and submitted to the variance analysis. The comparisons between the averages were made by the Scott-Knott test (p<<NUM>) (<NUM>), and the effectiveness of the treatments was calculated according to ABBOTT (<NUM>).

Treatments and form of application: The products in the study were used to treat soy seeds, cultivar NA <NUM> RG. For the treatment of the seeds, the application volume used was <NUM> of broth per <NUM> of seeds", to which water was added at the prescribed dose of the product, until the volume of <NUM> was reached. Then, <NUM> of soy seeds were added in a plastic bag together with <NUM> of the mixture (product + water). Said bag was closed and agitated until the products turned fully homogenized. A sample of approximately <NUM> grams of each treatment was removed and sent to Laboratério de Análises de Sementes credited by the Ministério da Agricultura Pecuária e Abastecimento - MAPA - for the germination tests. Soil and root samples were collected randomly from five distinct points of the useful portion that made out a compound sample, with approximately <NUM> of soil and <NUM> of root. The collections were carried out at a depth of <NUM>-<NUM>. For the quantification of eggs and younglets of P. brachyurus, the samples of each experimental portion were sent to the Nematology Laboratory so that P. brachyurus younglets and eggs could be quantified. The extration of the target in the soil was carried out using the centrifugal flotation technique in a sucrose solution proposed by JENKIS.

(<NUM>) in <NUM><NUM> of soil, wherein the evaluations had been made previously <NUM> and <NUM> days after sowing. For the quantification of nematodes in the root, the liquefier technique proposed by COOLEN & D'HERDE (<NUM>) was used in <NUM> of root, wherein the evaluations had been made <NUM> and <NUM> DAS. The reproduction factor (RF) was calculated, that is, the relationship between the final population (Fp) and the initial population (Ip) of nematodes, between <NUM> and <NUM> days after sowing, according to the formula proposed by OOSTENBRINK (<NUM>), where FR = Fp/Ip. When the plants bloomed (R3), the height of <NUM> plants in the center of each experimental portion was evaluated, by measuring the length of the stem from the soil level to the last leaf of the plant, with the aid of a measuring device. To estimate the productivity of the crop in kilograms per hectare, the grains were harvested and weighed in <NUM><NUM> and the humidity was set to <NUM>%.

In the evaluations of the height of plants (Table <NUM>), the applied treatments were not statistically differed from the standard, wherein the height of plants ranged from <NUM> to <NUM>.

In the evaluations of the effect of the applied treatments on the number of young and/or adult plants in <NUM><NUM> of soil (Table <NUM>), at the <NUM>th day after sowing no statistical differences between the standard and the evaluated doses of the biological formulation have been noticed. However, the product in the study has provided a control efficiency of up to <NUM>%, when compared with the standard. At the <NUM>th day after sowing, the biological mixture in doses of <NUM>, <NUM> and <NUM>. kg-<NUM> seeds had provided an efficient control of the population of nematodes in the soil with an efficiency ranging from <NUM>% to <NUM>%, statistically different from the standard treatments, and a higher dose of the biological formulation (<NUM> and <NUM>. kg-<NUM> seeds, respectively).

Table <NUM> shows the effect of the applied treatments on the number of young and/or adult plants in <NUM> of root. At the <NUM>th day after sowing, the biological formulation in doses of <NUM> and <NUM>. kg-<NUM> of seeds was superior to standard treatments, acting positively in the control of phytonematodes in lesions and exhibiting a control efficiency between <NUM> and <NUM>%, respectively. At the <NUM>th day after sowing, a higher reduction in the number of phytonematodes in the roots was observed, showing a significant difference between the standard treatments and the lower dose (<NUM>. kg-<NUM> seeds). Doses of <NUM>, <NUM>, <NUM> and <NUM>. kg-<NUM> seeds of the biological formulation showed efficiencies that ranged from <NUM>% to <NUM>% (Table <NUM>).

The high efficiency of the biological formulation in the control of phytonematodes in the roots occurs to the detriment of the characteristics of the microorganisms of the composition. Said microorganisms colonize the rhizosphere and reduce the penetration of nematodes into the roots, due to the action of nematicidal metabolites produced by bacteria, or the reduction of the chemical signalling exudated by the plants, that guide the nematodes towards the roots (SIDDIQUI & MAHMOOD, <NUM>).

The consequences of the control of phytonematode in lesions on the reproduction factor is presented in Table <NUM>. <NUM> and <NUM> after the sowing, the biological mixture in doses of <NUM> and <NUM>. kg-<NUM> seeds has promoted significantly the reduction of the reproduction factor, exhibiting an efficiency of <NUM>% and <NUM>% <NUM> and <NUM> days after the sowing, and <NUM>% and <NUM>% <NUM> days after sowing, respectively, indicating that at such doses (<NUM> and <NUM>. kg-<NUM> seeds) the product is efficient in preventing the population of nematodes in lesions from increasing significantly, both in the soil and in the roots.

As to the productivity of the culture, no statistical difference between the applied treatments and the standard has been noticed. However, the treatments with the biological formulation had provided increments of <NUM> to <NUM>% in the productivity of the crop (Table <NUM>). The same trend was observed in the evaluation of the weight of <NUM> grains, where no significant difference between the treatments has been noticed.

In view of the above, though significant effects on the promotion of the plant growth, productivity and weight of <NUM> grains have not been noticed, the biological mixture was efficient in the reduction of the population nematodes in root lesions, both in the soil and in the roots, and interfered negatively with the reproduction factor of same. The biological formulation can be included in programs for handling said nematode.

None of the evaluated treatments caused phytotoxicity to soy plants (Glycine max), cultivar NA <NUM> RR. The treatments using the biological formulation, when applied in doses of <NUM>, <NUM>, <NUM> and <NUM>. kg-<NUM> seeds, is efficient in the control of the population of nematodes in lesions in the soil by <NUM>% and in the roots by <NUM>%, and is also efficient in lowering the reproduction factor of nematodes by <NUM>% compared to the standard, and thus it can be considered as an efficient tool in the fight against nematode Pratylenchus brachyurus.

The preparation of the formulation for biological control of phytonematodes should follow the following flow of events:.

The present invention may be applied either by using plantation furrows, planting bars, treatment of seeds or even treatment of industrial seed by sowing machines for the control of phytonematodes. The application method will have to be analyzed case by case and will depend on the technical conditions and needs of each producer.

Claim 1:
COMPOSITION FOR BIOLOGICAL CONTROL OF PHYTONEMATODES, more precisely a composition with nematicidal effect in damage mitigation and control of phytonematodes on cultivable plants; characterized by consisting of microorganisms, in colony forming units, c.f.u./g at a concentration being:
Bacillus subtilis - <NUM> x <NUM><NUM> c.f.u./g;
Bacillus licheniformis - <NUM> x <NUM><NUM> c.f.u./g;
Bacillus amyloliquenfacies - <NUM> x <NUM><NUM> c.f.u./g;
at the following concentrations:
Bacillus subtilis - <NUM> to <NUM>%, by weight;
Bacillus licheniformis - <NUM> to <NUM>%, by weight;
Bacillus amyloliquefaciens - <NUM> to <NUM>%, by weight;
and
additives - <NUM> to <NUM>%, by weight, being selected from the group consisting of dispersants selected from the group consisting of water-soluble ionic polymers, water-soluble anionic polymers, and surfactants selected from the group consisting of anionic surfactants and non-ionic surfactants, and combinations thereof;
excipients - <NUM> to <NUM>%, by weight, being selected from the group consisting of silicas, talc, bentonite, carbohydrates, carbonates, milk , powder milk, and combinations thereof.