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WO2016165037A1 - Composition biologique de lutte contre les nématodes - Google Patents

Composition biologique de lutte contre les nématodes Download PDF

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Publication number
WO2016165037A1
WO2016165037A1 PCT/CL2016/050016 CL2016050016W WO2016165037A1 WO 2016165037 A1 WO2016165037 A1 WO 2016165037A1 CL 2016050016 W CL2016050016 W CL 2016050016W WO 2016165037 A1 WO2016165037 A1 WO 2016165037A1
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access code
composition
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rgm2155
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English (en)
Spanish (es)
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Erwin ABALLAY ESPINOZA
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Universidad de Chile
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Universidad de Chile
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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/13—Brevibacterium

Definitions

  • 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.
  • 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., Rotyienchu ⁇ us spp., Xiphinema spp. and Tyienchulus spp.
  • 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.
  • 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).
  • rhizobacteria that is, bacteria that naturally inhabit the roots or radical area of plants (that inhabit the rhizosphere).
  • 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.
  • 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 .
  • 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.
  • beneficial examples of plant growth preparation are indicated; but no analysis of the nematicidal properties of said strain is mentioned.
  • 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
  • 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 Bacil ⁇ us 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 Bacil ⁇ us weihenstephanensis (access code RGM2155) on the mobile states of the Tyienchulus nematode semipenetrans
  • 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), Bacil ⁇ us 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 Bacil ⁇ us 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.
  • 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.
  • 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.
  • isolated refers to the rhizobacterial strains of the present invention that have been removed and purified from their natural environment.
  • mutant 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.
  • 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.
  • 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.
  • 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.
  • 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, Me ⁇ oidogyne and Tylenchulus mainly, or other nematodes susceptible to be bio-controlled by the composition based on the bacteria Bacillus weihenstephanensis (access code RGM2155), Brevibacierium f ⁇ gorifolerans12 ) or a combination thereof in any of its variants, or binders of said strains with nematicidal activity.
  • Bacillus weihenstephanensis access code RGM2155
  • Brevibacierium f ⁇ gorifolerans12 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.
  • 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.
  • 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,
  • EXAMPLE 1 In vitro tests to evaluate the efficacy of the composition for the biological control of nematodes, s3 ⁇ 4 Evaluation of the nematicidal activity of the composition on the mortality of mobile states of 3 species of phytoparasite nematodes.
  • Meioidogyne ethiopica and Tylenchulus semipenetrans are also reflected in the graphs of Figures 1, 2 and 3, respectively.
  • 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 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 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.
  • EXAMPLE 2 Potted trials to evaluate effsca and composition for the biological control of nermatodes
  • 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).
  • 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,
  • composition for the biological control of neroá 'i3 ⁇ 4 Evaluation of the nematicidal activity of the composition on
  • 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).
  • Pf initial population

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  • Pest Control & Pesticides (AREA)
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Abstract

La présente invention concerne une composition nématicide et un procédé dans lequel on utilise ladite composition pour lutter contrer les nématodes pathogènes présents dans les plantes. Cette composition comprend les rhizobactéries Brevibacterium frigoritolerans, Bacillus weihenstephanensis et les éléments dérivés de leur culture, ou une combinaison de ceux-ci; ainsi qu'un support agronomiquement acceptable. Le procédé d'élimination des nématodes comprend l'application de ladite composition nématicide sur les terrains cultivés des plantes infectées.
PCT/CL2016/050016 2015-04-17 2016-04-11 Composition biologique de lutte contre les nématodes Ceased WO2016165037A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CL0996-2015 2015-04-17
CL2015000996A CL2015000996A1 (es) 2015-04-17 2015-04-17 Composición biológica para el control de nemátodos

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WO2016165037A1 true WO2016165037A1 (fr) 2016-10-20

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112322516A (zh) * 2020-05-19 2021-02-05 陕西省生物农业研究所 一种猕猴桃叶枯病新型生防菌剂的制备方法与应用
CN114350565A (zh) * 2022-01-24 2022-04-15 山东农业大学 一株耐寒短杆菌多功能菌株及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103952348A (zh) * 2014-04-24 2014-07-30 烟台地元生物科技有限公司 耐寒短杆菌及微生物菌剂和它们的应用
WO2014193746A1 (fr) * 2013-05-31 2014-12-04 Novozymes Bioag A/S Compositions et procédés pour l'activation de la germination

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014193746A1 (fr) * 2013-05-31 2014-12-04 Novozymes Bioag A/S Compositions et procédés pour l'activation de la germination
CN103952348A (zh) * 2014-04-24 2014-07-30 烟台地元生物科技有限公司 耐寒短杆菌及微生物菌剂和它们的应用

Non-Patent Citations (3)

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Title
CAMPOS, V. ET AL.: "Volatiles produced by interacting microorganisms potentially useful for the control of plant pathogens.", CIÊNCIA E AGROTECNOLOGIA, vol. 34, no. 3, 2010, pages 525 - 535, XP055025274 *
LI, M. ET AL.: "A strategy to discover potential nematicidal fumigants based on toxic volatiles from nematicidal bacteria.", AFRICAN JOURNAL OF MICROBIOLOGY RESEARCH., vol. 6, no. 31, 16 August 2012 (2012-08-16), pages 6106 - 6113, XP055321654 *
RAE, R. ET AL.: "A subset of naturally isolated Bacillus strains show extreme virulence to the free-living nematodes Caenorhabditis elegans and Pristionchus pacificus.", ENVIRONMENTAL MICROBIOLOGY, vol. 12, no. 11, 2010, pages 3007 - 3021, XP055321657 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112322516A (zh) * 2020-05-19 2021-02-05 陕西省生物农业研究所 一种猕猴桃叶枯病新型生防菌剂的制备方法与应用
CN114350565A (zh) * 2022-01-24 2022-04-15 山东农业大学 一株耐寒短杆菌多功能菌株及其应用
CN114350565B (zh) * 2022-01-24 2023-06-09 山东农业大学 一株耐寒短杆菌多功能菌株及其应用

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