WO2016165037A1 - Biological composition for controlling nematodes - Google Patents

Abstract

The present invention provides a nematicidal composition and a method that uses said composition to control nematode pathogens present in plants. The composition comprises the rhizobacteria Brevibacterium frigoritolerans and Bacillus weihenstephanensis and elements derived from the culture thereof, or a combination of these, and an agriculturally acceptable carrier. The method for eliminating nematodes comprises applying the nematicidal composition on croplands containing the infected plants.

Description

BIOLOGICAL COMPOSITION FOR NEMATE CONTROL

The present invention relates to the technical field of agriculture, specifically with biocides, and in particular provides a biological composition for the control of pathogenic nematodes present in plants.

Background of the invention

Plant parasitic nematodes are a major current problem for agriculture worldwide. More than 4,100 species of phytoparasite nematodes have been identified that affect various types of crops in different territories and soil conditions (Nicoi JM, Turner SJ, Coyne DL, den Nijs L, Hockland S, Tahna Maafi Z, Current Nematode Threats to World Agricuiture Chapter 2. Genomics and Molecular Genetics of Piant-Nematode interactions 201 1) Among the most harmful nematodes to plants of commercial interest are species of the genera Meloidogyne spp., RotyienchuÍus spp., Xiphinema spp. and Tyienchulus spp. (Luc, M., Sikora, RA., Bridge, J. (Eds.). Píant parasitic nematodes in subtropical and tropical agriculture. 2005. Cabi. Second edition). Phytoparasite nematodes are found within the most widely distributed pests, whose eradication is often very difficult and expensive. It is estimated that crop losses from nematodes correspond to 14.6% and 8.8% in underdeveloped and developing countries, respectively (Nicol JM, Turner SJ, Coyne DL, den Nijs L, Hockland S, Tahna Maafi Z. Current Nematode Threats to World Agriculture Chapter 2. Genomics and Molecular Genetics of Plant-Nematode Interactions 201 1).

Different strategies have been developed for the control and elimination of phytoparasite nematodes. One of these strategies includes the use of nematicidal products derived from non-phytotoxic chemical compounds, such as halogenated fumigants, organophosphates and carbamates. The disadvantage of using these chemical nematicides is that they are highly dangerous to health. human cause poisoning and potential mortality (World Health Organizer !. (1990). Public health impact of pesticides used in agricultuiture: p.88.). Some countries have already banned their use due to the presence of these toxins in groundwater and drinking water, and toxic traces in plants and fruits. Consequently, these compounds must be applied with caution and for this they require specialized personnel and machinery in order to avoid accidents, which increases costs (Gowen SR. Chemical control of nematodes: efficiency and side-effects. FAO Plant Production and Profection Paper - 144. Rome 1997). As a result of the above, the development of new nematicides has advanced towards obtaining products of natural and / or biological origin.

Nematicides of biological origin seek to enhance the natural enemies of nematodes, using fungi or bacteria capable of killing nematodes through parasitism, opportunism or production of toxins or metabolites. In the control of phytoparasite nematodes, fungal species of the genera Arthrobotrys spp., Fusarium spp, and Monacrosporium spp. as shown in patent application WO2012140207, as well as bacterial species such as Baciiius megaterium (WO20141 18401), Baciiius subíiiis (WO2013178666), Baciiius firmus (WO2014043058), Pseudomonas aeruginosa and Pseudomonas polished (WO2014086udse).

Baciiius weihenstephanensis and Brevibacterium frigoritoierans bacteria are species classified within the group of rhizobacteria, that is, bacteria that naturally inhabit the roots or radical area of plants (that inhabit the rhizosphere). In the publication ZhiFang, L., YingQi, G., íngHe, M., YueQiu, H. Optirnization of the culture condition for Baciiius weihenstephanensis strain ¡V1C67 producing volatile nematicidai. 2009. Chínese Journal of Biological! Control, 25: 4, 339-347, the optimization of the culture conditions of the bacterium Baciiius weihenstephanensis (strain MC67) is described and a nematicidal activity is observed on the Bursaphelenchus xyiophiíus nematode, which parasites the pine wood and not its estate. On the other hand, there is an in vitro study that individually analyzes the nematicidal effect of Bacillus weihenstephanensis isolated from German soil in Caenorhabditis e! Egans and Pristionchus pacificus (Rae, R., Atsenko, I., Witte, H., Sommer, RJ A subset of naturaily isoiated Bacillus strains show extreme virulence to the free-living nematodes Caenorhabditis eiegans and Pristionchus pacificus. 2010. Environmentai microbioiogy, 12. 11: 3007-3021), however these nematodes are not phytopathogens, that is, their cycle vital does not include infection of plants at any stage (Dieterich, C, Clifton, SW., Schuster, LN ,, Cbinwalla, A ,, Deiehaunty, K., Dinkelacker,!., Sommer, RJ. The Pristionchus pacificus genome provides a unique perspective on nematode lifestyle and parasitism 2008. Nature genetics, 40.10: 1 193-1 198) and have no economic interest.

Within the nematicidal biological formulations existing in the state of the art, patent application CN103952348 mentions a microbial inoculum containing only the Chinese strain of Brevibacterium frigoritolerans (CGMCC No. 8837), whose nematicidal activity was evaluated only in the unknown Meloidogyne nematode . On the other hand, the granted patent CN102229900 discloses a probiotic preparation to be used as a fertilizer and a method for its preparation that includes an inoculum of Bacillus weihenstephanensis, among others. In this last document, beneficial examples of plant growth preparation are indicated; but no analysis of the nematicidal properties of said strain is mentioned.

Although there have been multiple efforts and combinations of agents for the control of nematodes, to date it has not been possible to obtain an effective nematicidal composition against the nematodes that affect plants of economic interest, especially their roots, and that is easy to use. and friendly to the environment.

Summary of the Invention

An object of the present invention relates to strains of isolated and purified rhizobacteria corresponding to the species Brevibacterium frigoritolerans with the access code RGM2156 and Bacillus weihenstephanensis with the access code RGM2155, or mules thereof with nematicidal activity.

A second object of the present invention relates to a biocidal composition comprising at least one biological control agent selected from the group consisting of: the rhizobacterium of the strain Brevibacterium frigorito! Erans (access code RGM2156), the rhizobacterium of the strain Bacillus weihenstephanensis (access code RG! Vl 2155), at least one mule of these strains that maintains the essential identification characteristics of their parental strain, at least one metabolite produced by the culture of said bacteria or mutants, a supernatant obtained from the culture of said bacteria or mutants, an isolate of said strains or mutants thereof, or a combination of the above agents; and an agronomically acceptable carrier. Said biological control agents possess nematicidal activity.

Each rhizobacterial strain is in the concentration range between 1 x 10 ⁶ and 1 x 10 ⁸ CFU per gram of composition. The combination of biological control agents is a mixture of the strains of rhizobacteria Brevibacterium frigoritolerans (access code RGM2156) and Baciiius weihenstephanensis (access code RGM2155), as they are found in the composition in a preferred embodiment of invention in a 1: 1 ratio.

Another object of the present invention relates to a method for the biological control of phytoparasite nematodes, which comprises providing the composition previously described in any of its variants, and applying it to a crop land of a plant of interest by means of a technique. appropriate such as ground injection, drip irrigation, sprinkling, spraying, manual irrigation, direct application in the form of powder, granules, capsules, botos or a combination of yourself.

Figure 1 is a graph with the results of the evaluation of the nematicidal effect of the culture supernatants of the rhizobacteria Brevibacterium frigoritolerans (access code RG 2156) and Baciiius weihenstephanensis (access code RGM2155) on the mobile states of the Xiphinema index nematode.

Figure 2 is a graph with the results of the evaluation of the nematicidal effect of the culture supernatants of the rhizobacteria Brevibacterium frigoritolerans (access code RG 2156) and Bacilíus weihenstephanensis (access code RGM2155) on the mobile states of the nematode Meloidogyne ethiopica.

Figure 3 is a graph with the results of the evaluation of the nematicidal effect of the culture supernatants of the rhizobacteria Brevibacterium frigoritolerans (access code RGM2156) and Bacilíus weihenstephanensis (access code RGM2155) on the mobile states of the Tyienchulus nematode semipenetrans

Detailed description of the invention

The present invention relates to a biocidal composition comprising biological control agents with nematicidal activity and a method for e! Biological control of phytoparasitic nematodes that uses said nematicidal composition, to which it is applied on fields of crops infected with these pathogens, so that the composition accesses the roots of the plants, where the nematodes are preferentially settled. The nematicidal composition comprises an inoculum of the cultivation of rhizobacteria isolated from the soil of the central zone of Chile Brevibacterium frigoritolerans (access code RGM2156), Bacilíus weihenstephanensis (access code RGM2155) or a combination thereof; or the supernatants, metabolites or isolates obtained by the culture of said bacteria, or a combination of the mentioned agents. The invention may also comprise any mutant strain derived from the rhizobacteria Brevibacterium frigoritolerans (access code RGM2156) or Bacilíus weihenstephanensis (access code RGM2155) that possesses the essential identification characteristics of the mentioned rhizobacterial strains and that possesses nematicidal activity. Both strains were deposited in the Bank of Microbial Genetic Resources of the INIA (Institute of Agricultural Research) - Chile with the previously referenced access codes.

The nematicidal composition of the invention additionally contains an agronomically acceptable carrier that allows its proper application. The composition may have a liquid or powder format, depending on the carrier used. The microorganisms added to the composition are in the range between 1 x 10 ⁶ - 1 x 10 ⁸ CFU per gram of composition. In a preferred embodiment the composition comprises mixing the strains of rhizobacteria Brevibacterium frigoritoierans (access code RGIV12156) and Bacillus weihenstephanensis (access code RG1V12155) or the supernatants of their culture in a 1: 1 ratio.

In the context of the present invention, "biological control" means the regulation of the number of pathogens or parasites of plants through the use of a second biological organism belonging to the group of rhizobacteria or agents derived therefrom, such as supernatant of its culture, the metabolites produced by it or a lysate of said culture, so that they are equally effective in attacking the pathogen and / or affecting its survival.

The term "isolated" refers to the rhizobacterial strains of the present invention that have been removed and purified from their natural environment.

The term "mutant", as used in the present invention, refers to a variant of the parental bacterial strain that possesses any change in the DNA sequence and a nematicidal activity greater than or equal to the parental strain.

The term "parental strain" refers to each strain originally deposited in the Microbial Genetic Resources Bank of the NIA (Chile) before being modified by any appropriate technique. To obtain mutant strains from the parental strains disclosed in the present invention, various methods of conventional mutagenesis can be used, such as treatment with nitrosoguanidine, ethyl methanesuifonate, radiation gamma, ultraviolet radiation, x-rays or other techniques widely known in the state of the art. Genetic engineering techniques can also be used, which are known by any person skilled in the art, to obtain mutant strains with improved nernaticidal activity or another type of advantage that allows its convenient application for the control of phytoparasites.

The term "metabolite" refers to any compound, substance or by-product of the rhizobacterial culture object of the present invention that possesses nematicidal activity. The term "supernatant" refers to the fluid conditioned by the cultivation of the rhizobacteria object of the present invention once it has been separated from the bacteria.

The term "iisado" refers to the fluid that contains bacterial cells that have been previously cultured or form a suspension, and that are destroyed by mechanisms that compromise the integrity of the cell, which are widely known in the state of the art.

"Essential identification characteristics" means those properties that have the isolated rhizobacteria object of the present invention. In particular, the characteristic properties of Brevibacterium frigoritolerans are described for example in the publication Selvakumar, G., Sushii, S. NL, Stanley, J., Mohan, M., Deol, A., Rai, D., ... & Gupta, HS (201 1). Brevibacterium frigoritolerans a novel entomopathogen of Anómala dimidiata and Holotrichia iongipennis (Scarabaeidae: Coleóptera). Biocontroi Science and Technology, 21 (7), 821-827. As a second example, in the publication Thorsen, L, Hansen, BM, IMielsen, KF, Hendriksen, NB, Phipps, RK, & Budde, B. B, (2006). Characterization of emetic Baciiius weihenstephanensis, a new cereulide-producing bacterium. Applied and environmentai microbioiogy, 72 (7), 51 18-5121 describes the characteristics that identify Baciiius weihenstephanensis. A second object of the invention is a method for the control of phytoparasite nematodes in cultures, which comprises the application of the Nematicidal composition previously mentioned in any of its variants in the crop field to reach the roots of the plants. The application of the composition to the ground can be carried out by various techniques also known, such as: ground injection, drip irrigation, spraying, spraying, manual irrigation, direct application in the form of powder, granules, capsules, bowling or a combination of themselves. The composition can be applied to plants of any age, young or adult, whether they are planted or in the process of transplantation, by immersing their roots in the composition in any of its variants. The composition can be used on any soil, such as in massive farmland, greenhouses, pots, fireworks and gardens. The crops of interest where the unrestricted composition of any kind can be used are for example: vine, olive, mango, tomato, papaya, kiwi, pistachio, banana, blueberries and citrus fruit trees and in general in any cultivation of susceptible plants of being infected by the nematodes belonging to the genera Xiphinema, Meíoidogyne and Tylenchulus mainly, or other nematodes susceptible to be bio-controlled by the composition based on the bacteria Bacillus weihenstephanensis (access code RGM2155), Brevibacierium fñgorifolerans12 ) or a combination thereof in any of its variants, or binders of said strains with nematicidal activity.

The form of administration of the composition may be dry, as a powder, capsules, granules, boluses or tablets, or as a liquid, as a solution, or in the form of foam, gels, emulsions or any other acceptable form in agriculture . The final formulations may also contain agronomically acceptable excipients, such as disintegrating agents and / or binders, wetting agents, surfactants and stabilizers.

The composition in any of its variants is prepared according to conventional procedures and formulations known in the state of the agricultural-chemical art. Granular or powder compositions are usually dispersed in solid acceptable agronomic carriers such as clay, kaolinite powder, volcanic stone, diatomaceous earth, silicates, flour wood, Fuüer land, plaster, calcite powder or the like, among others. Liquid compositions are usually dispersed or diluted in acceptable agronomic liquid carriers such as water or organic solvents (aromatic compounds, terpenics or ketones), among others. Optionally the composition may include chemical agents attracting nematodes and / or phytohormones or additional metabolites to promote healthy plant growth.

The present invention provides an alternative to commonly used chemical nematicides and to nematicidal compositions based on known biological controllers, through a composition based on strains of specific rhizobacteria for the control of phytopathogenic nematodes or mixtures thereof, with demonstrated efficiency. The nematicidal composition is effective in controlling a wide range of species and genera of nematodes, in various types of crops and soils. Additionally, the composition of the invention promotes the root growth of plants, facilitating their healthy development,

Examples of embodiment of the invention are presented below, which have been included for the purpose of illustrating the invention, its preferred embodiments and comparative examples, but in no case should they be considered to restrict the scope of the patent application, which only It is delimited by the content of the claims attached here.

EXAMPLES

EXAMPLE 1: In vitro tests to evaluate the efficacy of the composition for the biological control of nematodes, s¾ Evaluation of the nematicidal activity of the composition on the mortality of mobile states of 3 species of phytoparasite nematodes.

To carry out the evaluation of the nematicidal activity of the composition of the invention on the mortality of mobile states of the phytoparasitic nematodes Xiphinema index, Meioidogyne ethiopica and Tylenchulus semipenetrans, the rhizobacteria Brevibacterium frigoritolerans (access code RGM2156) and Baciilushens code of access RGM2155) were grown in a medium containing tryptic soy broth (TSB, from the acronym Tryptic Soy Broth) 15 g / L at 22 ° C for 44 hours on a shaker at 180 rpm, until reaching a concentration of 1 x 10 ⁶ colony forming units CFU / mL After carrying out the culture of the bacteria, the suspension was centrifuged twice at 4000 rpm for 20 minutes and the supernatant was collected and in this a 50 nematode immersion was carried out in mobile states for 48 and 72 hours, after which the mortality of the nematodes was determined. Those immobile individuals were considered dead, even after pricking them with a needle. The effect of the supernatants of both strains was determined separately and in a mixture (in a 1: 1 ratio). A study was conducted for each of the three nematodes mentioned above under laboratory conditions. As a control treatment, untreated nematodes present in the same TSB nutrient solution used for bacterial culture were used. Tables 1, 2 and 3 show the results of mortality tests on mobile states of iodex Xiphinema nematodes,

Meioidogyne ethiopica and Tylenchulus semipenetrans, respectively, which are also reflected in the graphs of Figures 1, 2 and 3, respectively.

Table 1. Nematicidal effect of the rhizobacterial culture supernatant on mobile states of Xiphinema index.

{%) / Weather

Treatments

 48h 72h

 Brevibacterium frígoritolerans (code of.

 53.7 b access RGM2158)

Bacilíus weihenstephanensis (code ₄ „_.

 80.5 b access RGM2155) '

 B, frígoritolerans (access code

 RGIV12156) + B. weihenstephanensis 54.04 b 88.61 b (access code RGM2155) (1: 1)

 Control 3.25 to 9.46 a

The data correspond to the means of four repetitions. Different letters in the same column are statistically different from the control treatment according to the Dunnet test p <0.05. Table 2. Nematicidal effect of the supernatant and of the rhizobia culture on mobile states of Meioiá ogyne ethiopica.

Mortality (%) / Time

Treatments

 48h 72h

 Brevibacterium frígoritoíerans (code of

 3.2 to 53 b access RG1V12156)

 Badil us weihenstephanensis (code of

 41.5 b 54.2 b access RGM2155)

 B. frígoritoíerans (access code

 RGM2158) + B. weihenstephanensis 30.71 b 70.51 b

(access code RG1V12155) (1: 1)

 Control 2, 18 to 3, 10 to

The data correspond to the means of four repetitions. Different letters in the same column are statistically different from the control treatment according to the Dunnet test p <0.05.

Table 3. Nematicidal effect of the rhizobacterial culture supernatant on mobile states of Tyienchuius semipenetrans.

Treatments

 Brevibacterium frígoritoíerans (code of

 12. 5 3 42.8 b access RGM2158)

 Baciilus weihenstephanensis (code of

 10.2 to 25.3 b access RGM2155)

 B, frígoritoíerans (access code

 RGM2156) + B. weihenstephanensis 20.24 b 71, 12 c

(access code RGM2155) (1: 1)

 Control 9.50 to 1 1, 73 to

The data correspond to the means of four repetitions. Different letters in the same column are statistically different from the control treatment according to the Dunnet test p <0.05.

The results of these tests show that the nematicidal composition formed by the mixture of the supernatants of the crop of rhizobacteria Brevibacterium frígoritoíerans (access code RG1V12156) and Baciilus weihenstephanensis (access code RGM2155) is highly efficient since it produces a high percentage of mortality over the three species of nematodes analyzed, which is statistically significant compared to the untreated control, as shown in Tables 1, 2 and 3. In addition it was found that the use of the mixture of the supernatants of the culture of the strains Brevibacterium frigorito! erans (access code RGM2156) and Baciüus weihenstephanensis (access code RGM2155) increases the range of action of the nematicidal composition since it allows obtaining a high percentage of mortality on the analyzed nematodes, which is greater than obtained by the supernatants of each bacterium separately. These differences are even significant as shown in the 72-hour treatments on the Tylenchulus semipenetrans nematode in Table 3. These results demonstrate that the nematicidal composition allows synergistically to broaden the spectrum of the target nematodes.

The species studied are very frequent in Chilean vineyards and fruit trees. The first two species are considered as representative of other species of the same genera, such as X. americanum s. /., M. incognita, M. javanica or M. hapia, hence this study allows extrapolating the results obtained with the Composition of the invention to other phytopathogenic nematodes.

^" ú) Evaluation of the nematicidal activity of the composition on the

To determine the nematicidal activity of the composition on the hatching of eggs of the phytoparasitic nematode Meioidogyne ethiopica, approximately 50 eggs were incubated with 3 mL of the culture supernatants of said strains in 5 mL Petri dishes for 48 hours and subsequently the percentage of hatching. As seen in Table 4, treatment with the culture supernatants of the rhizobacteria mentioned significantly decreases the hatching percentage of M. ethiopica eggs compared to the untreated control condition in TSB nutrient solution.

Table 4. Nematicidal effect of the rhizobacterial culture supernatant on eggs of Meioidogyne ethiopica.

Treatment

 Hatching (%} (bacterial isolation)

Brevibacterium frigoritoierans (access code RG 2158) 15.0 ^* ± 2.2 Bacii! Us weihenstephanensis (access code RGM2155)

 Control (TSB medium) 90, 1 ± 4.8

^* : p <0.05 according to Dunnett's test, n = 6.

EXAMPLE 2: Potted trials to evaluate effsca and composition for the biological control of nermatodes

Matemat¡aaa of the composition about

To evaluate the effect of the nernaticidal composition comprising each strain of the rhizobacteria Brevihacierium frigoritolerans (access code RG 2156) and Bacillus weihenstephanensis (access code RG 2155) separately, tests were carried out with 8 vine plants var. Two-month-old Thompson Seedless for each bacterium and control in potted plants under greenhouse conditions. These plants were grown from stakes in 0.5 L pots with sterile peruta substrate, subsequently removed from these containers, the radical system was submerged in the bacterial suspension at a concentration of 1 x 10 ⁶ CFU / mL, established in the definitive 5 L pots and inoculated with 400 specimens of the Xiphinema index nematode per pot. Each pot was then watered with the same bacterial suspension, with 100 mL per pot. After 6 months the plants were removed from the pots, the population density of live nernátodos in the substrate was measured after the treatments and the aerial vegetal mass. Then the roots were washed and their mass and the number of damages per gram of root were measured. For this purpose, the number of apical nodules present in the roots was determined by means of a magnifying glass, which is the symptom caused by the nerves when feeding. The results are shown in Table 5, where it is observed that the plants treated with each strain of rhizobacteria have significantly less damage to their roots and a greater aerial and radical mass compared to the control (only nermatodes). Furthermore, it is observed that the treatments with the strains of rhizobacteria mentioned significantly reduce the number of live nernatates present in each pot. Table 5. Nematicidal effect of rhizobacterial strains on vines infested with 400 copies of Xiphinema index.

: p <0.01; ^* : p <0.05 (according to the Dunnetf test) indicate differences compared to control 2. n = 4. Subsequently, a study was carried out with free plants of var.

Thompson Seedless 3 months developed in 5 L pots grown this time with agricultural soil and inoculated with 1000 specimens of the Xiphinema index index nematode per pot. This study lasted 2 seasons (20 months) and was carried out in parallel with a second trial under the same conditions in order to analyze the efficacy of strains of rhizobacteria. After this period, the damage to the plant was evaluated by counting the root gills generated by the infection of said nematode. The results are shown in Table 6, where it is observed that the treatments with the mentioned rhizobacterial strains reduce the number of gills of the roots of the plants produced by the nematode infection compared to the infested and untreated plants (control ). Table 6. Nematicidal effect of rhizobacterial strains on vines infested with 1000 specimens of Xiphinema index.

^* : p <0.05 (according to Dunnett's test) indicates differences compared to the control, n = 6 for each trial. U) Evaluation of the nematicidal activity of the composition on

Greenhouse cultures of table grape plants of 2 months old var. Chardonnay in 5 L pots filled with naturally infested soil with phytoparasite nematodes. The bare root plants were inoculated for one minute with suspensions of the strains Brevibacterium frigoritoierans (access code RGM2156) and Bacillus weihenstephanensis (access code RGM2155) at a concentration of 1 x 10 ⁶ CFU / mL in an isotonic solution of MgS0 ₄ . After 5 months of study, the reproductive index of the Meloidogyne ethiopica nematode was evaluated, for which the ratio between the population or number of nematodes present at the beginning (Pi) and at the end of the study (Pf) was calculated. The results indicate that the strains used in the treatments are effective in reducing the population of the phytoparasite nematode studied, decreasing their reproductive index compared to the absolute control (plants without nematode control treatment, only received water irrigation as well as the remaining treatments ), being even more effective than the phosphor chemical contact nematicide used (Cadusafos - Rugby 200 CS in doses equivalent to 15 L of commercial product per hectare), as shown in Table 7,

Table 7. Nematicidal effect of rhizobacterial strains on vines naturally infested with Meloidogyne ethiopica.

 p <0.05 indicates significant differences with the absolute control according to Dunnett's test n = 6.

EXAMPLE 3: Field studies for

 composition for the biological control of neroá 'i¾ Evaluation of the nematicidal activity of the composition on

A field study was conducted in the town of Casablanca, V region, Chile, on vine plants var. 6 month Syrah. The radical systems of the plants in winter recess were inoculated prior to their establishment in a vineyard, in a replanting sector, by immersion in a suspension of the strains Brevibacterium frigoritoíerans (access code RG1V12156) and Bacilius weihenstephanensis (access code RGM2155 ) at a concentration of 1 x 10 ⁶ CFU / mL of running water and subsequently planted in clay loam soil with drip irrigation. The treatments were distributed randomly, using plots of 4 plants, 4 m ² , with 4 repetitions for each treatment. The natural populations of dominant phytoparasitic nematodes in such arable land belong to the genera Pratyienchus, Paratyienchus and Tylenchorhynchus. After the course of 15 months after planting the number of nematodes present in a sample of 250 cm ³ of soil (final population, Pf) was evaluated, which was compared with the initial number of nematodes (Pi) and the rate was calculated Reproductive of themselves (Pf / Pi). In the Table 8 shows that the plants treated with the suspensions of the nzobacterial strains mentioned showed a significant decrease in the reproductive rate of the nematode populations present in the cultivation land compared to the control plants that did not receive any treatment.

Table 8. Nematicidal effect of rhizobacterial strains on vines naturally infested with nematodes of the genera Pratylenchus, Paratylenchus and

 Tylenchorhynchus

 p <0.05 according to the Fisher minimum significant differences test n = 4.

Claims

one . An isolated and purified rhizobacterium of the genus Bacillus, CHARACTERIZED because it is the strain of the species Bacillus weihenstephanensis with the access code RGM2155, or a mutant thereof with nematicidal activity. 2. An isolated and purified rhizobacterium of the genus Brevibacteríum, CHARACTERIZED because it is the strain of the species Brevibacteríum frigoritolerans with the access code RGM2156, or a mutant thereof with nematicidal activity. 3. A biocidal composition, CHARACTERIZED because it comprises at least one biological control agent selected from the group consisting of: - the rhizobacterium of the strain Brevibacteríum frigoritolerans (access code RGM2156); - the rhizobacterium of the strain Bacillus weihenstephanensis (access code RGM2155); - at least one mutant of these strains with the essential identification characteristics of their parental strain; - at least one metabolite produced by the culture of said bacteria or mutants; - a supernatant obtained from the culture of said bacteria or mutants; - a lysate of said bacteria or mutants; - or combinations of the above agents; and an agronomically acceptable carrier. 4. The composition according to claim 3, CHARACTERIZED in that said biological control agent possesses nematicidal activity. 5. The composition according to claim 3, CHARACTERIZED in that said rhizobacteria of the strains Brevibacteríum frigorítolerans (access code RGM2156) and Bacillus weihenstephanensis (access code RGM2155) are in the concentration range between 1 x 10 ⁶ and 1 x 10 ⁸ CFU per gram of composition. 6. The composition according to claim 3, CHARACTERIZED in that said combination of biological control agents is a mixture of the strains Brevibacteríum frigorítolerans (access code RGM2156) and Bacillus weihenstephanensis of (access code RGM2155). 7. The composition according to claim 6, CHARACTERIZED in that the mixture of the strains contains said strains in a 1: 1 ratio. 8. A method for the biological control of CHARACTERIZED phytoparasite nematodes because it comprises the following steps: i) providing a composition comprising at least one biological control agent selected from the group consisting of: - the rhizobacterium of the strain Brevibacteríum frigorítolerans (access code RGM2156); - the rhizobacterium of the strain Bacillus weihenstephanensis (access code RGM2155); - at least one mutant of these strains with the essential identification characteristics of their parental strain; - at least one metabolite produced by the culture of said bacteria or mutants; - a supernatant obtained from the culture of said bacteria or mutants; - a lysate of said bacteria or mutants; - or combinations of the above agents; and an agronomically acceptable carrier; and ii) apply said composition to a crop land of a plant of interest. 9. The method according to claim 8, CHARACTERIZED in that the composition is applied by a technique selected from the group consisting of: direct injection to the ground, drip irrigation, sprinkling, spraying, manual irrigation, direct application in powder form, granules, capsules, bowling or a combination thereof.