WO2019160399A1 - Consorcio de bacterias resistentes a tiodicarb (carbamato) y a bifentrina (piretroide) y su uso en abonos líquidos. - Google Patents
Consorcio de bacterias resistentes a tiodicarb (carbamato) y a bifentrina (piretroide) y su uso en abonos líquidos. Download PDFInfo
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- WO2019160399A1 WO2019160399A1 PCT/MX2019/000005 MX2019000005W WO2019160399A1 WO 2019160399 A1 WO2019160399 A1 WO 2019160399A1 MX 2019000005 W MX2019000005 W MX 2019000005W WO 2019160399 A1 WO2019160399 A1 WO 2019160399A1
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- thiodicarb
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- 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
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- 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
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- 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
- A01N63/23—B. thuringiensis
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- 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
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- 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
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- 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
- C12N1/36—Adaptation or attenuation of cells
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- 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
Definitions
- NOVELTY consists in developing a consortium with specific bacterial species, adapted to resist, tolerate and mineralize the organic matter contaminated with carbamates such as thiodicarb and pyrethroids such as biphentrine, compounds that are currently used in the agricultural industry, livestock and present in organic waste.
- liquid BioAbonos are produced;
- This microbial consortium enriches them and a product with a bacterial microload is obtained that when applied to crops, captures atmospheric nitrogen and makes it available to plants, solubilizes the phosphorus present in soils such as P2O4 by adding an O to convert it into P2O5 assimilable by
- the plants contain antagonistic microorganisms of some pathogens, and repopulate the soil with beneficial bacteria. It is important to note that the microorganisms that make up our consortia ARE NOT GENETICALLY MODIFIED nor are they pathogenic for humans, or for animals, or for plants.
- Carbamates are insecticidal substances formed by an N atom attached to a labile group, carbamic acid; its main characteristic is its high toxicity, its low chemical stability and its zero accumulation in tissues; and pyrethroids, are molecules with insecticidal activity, remain longer in the environment since the chemical modification of their formula makes them more stable to sunlight and heat. These products are used indiscriminately in the agricultural and livestock industry, generating residuality.
- Pesticides are designed to kill, reduce or repel insects, herbs, rodents, fungi and other organisms that may threaten public health and the economies of nations. When these chemicals are handled or deposited improperly they can affect human health. ""The main risks related to human health from chronic low dose exposure are related to the occurrence of cancer, birth defects, nervous system conditions and the functioning of the endocrine system.” Assertions taken from: Childhood Pesticides Poisoning: Information for Advocacy and Action ", UNEP Chemicals, May 2004. In the development of our scientific research, we have found soils that have no micro-load due to the excessive use of agrochemicals to which they have been subjected .
- strains undergo several stages of stress through induced changes, adding traces to achieve chemo resistance to the synthetic pyrethroid biphentrine (2- methylbiphenyl-3-ylmethyl (2) - (1RS, 3RS) -3- (2-chloro 3, 3 , 3- trifluroprop-enyl) -2,2-dimethylcyclopropanecarboxylate;
- each strain was isolated and independently selected by successive stage cultures with increases in the concentration of synthetic pyrethroids such as biphentrine and carbamates such as thiodicarb.
- the percentages of each microorganism belonging to the consortium range between 20-30% of CFU of each of the
- strains The variability of the amount of each strain will depend on the characteristics of the substrate that the consortium will face.
- the required amount of the SPC Agar, Malta Agar (AM) and (PDA) culture medium is prepared to have viable colonies.
- the lethal dose (DL) for application to one hectare of both thiodicarb and biphentrine is determined.
- 10 boxes of Petri are prepared with 0.1 DL of thiodicarb or biphentrine. The boxes are initially incubated at a temperature of 25 ° C for 72 hours and in cold light.
- Tukey test is performed, at 5% probability.
- stages E-l and E-2 Stage 1 and Stage 2
- stage 2 the same concentration of thiodicarb is used to reinforce the growth of resistant strains).
- the resistant strains are transferred to solid culture media with 0.2 DL (0.000035 gr) of thiodicarb (E-3 and E-4, again in E-4 the same concentration of thiodicarb is used to reinforce the growth of resistant strains) presenting mortalities between 66 to 62%.
- stage E-4 organisms have been selected for 164 days.
- Stages E-5 and E-6 are subjected to a first addition of biphentrine; the culture medium is added with 0.1 DL of biphentrine (0.00000006 gr), presenting mortality of 62 to 61% (in the same way as the previous case, E-6 is used to reinforce the growth of resistant strains). Stages E-7 and E-8 are increased to 0.2 DL of biphentrine (0.00000012 gr) presenting 60% mortalities. At the end of E-8 (step of strengthening the growth of resistant strains) the adaptation process reaches
- Stages E-9 and E-10 0.3 DL (0.000053 gr) of thiodicarb is added, presenting mortality of 55 to 54%. (As in the previous stages, the E-10 is for growth reinforcement of resistant strains). In stages E-ll and E-12, 0.4 DL (0.000070 gr) of thiodicarb is added, presenting mortality of 52 to 48%. At the end of stage E-12 (step of strengthening the growth of resistant strains) 525 days of selection process are reached.
- stages E-13 and E-14 0.3 DL (0.00000018 gr) of biphentrine is added, presenting mortality of 52 to 48% (As in the previous stages, E-14 is for reinforcement of growth of resistant strains).
- stages E-15 and E-16 it is increased to 0.4 DL (0.00000024 gr) presenting 45% mortalities.
- stage 16 step of strengthening the growth of resistant strains 678 days of selection process are reached.
- stages E-17 and E-18 0.5 DL (0.000087 gr) of thiodicarb is added, presenting mortality of 42 to 40% (As in the previous stages, E-18 is for reinforcement of growth of resistant strains).
- stage E-19 and E-20 0.6 DL (0.00011 gr) of thiodicarb is added, presenting
- E-21 and E-22 are added 0.5 DL (0.00000030 gr) of? biphentrine, presenting mortality of 30 to 28% (As in the previous stages, the E-22 is for reinforcement of growth of resistant strains).
- E-23 and E-24 it is increased to 0.6 DL of biphentrine (0.00000036 gr), presenting mortalities between 25 and 23%.
- E-24 stage of strengthening the growth of resistant strains
- stages E-25 and E-26 0.7 DL (0.00012 gr) of thiodicarb is added, presenting mortality of 25 to 22% (As in the previous stages, E-26 is for reinforcement of growth of resistant strains).
- E-27 and E-28 0.8 DL (0.00014 gr) of thiodicarb are added, with mortalities of 20 to 18%.
- E-28 step of strengthening the growth of resistant strains
- E-30 is for reinforcement of growth of resistant strains.
- E-31 and E-32 it increases to 0.8 DL of biphentrine (0.00000042 gr), presenting mortality of 13 to 10%.
- E-32 step of strengthening the growth of resistant strains 1131 work days are reached.
- stages E-33 and E-34 the 0.9 DL (0.00016 gr) of thiodicarb, presenting mortality of 10% (As in the previous stages the E-34 is for reinforcement of growth of resistant strains).
- stages E-35 and E-36 1 DL (0.00018 gr) of thiodicarb is added, with mortalities of 9 to 8%.
- E-36 step of strengthening the growth of resistant strains
- E-37 and E-38 add 0.9 DL of biphentrine (0.00000042 gr); presenting 10% mortality in the 2 stages, (As in the previous stages the E-38 is for reinforcement of resistant strain growth).
- the E-39 and E-40 is increased to 1 DL of biphentrine (0.00000048 gr) presenting a mortality in 8 to 7% At the end of the 40 stage we have 1278 work days.
- microorganism is already resistant to traces of 1 DL of biphentrine and 1DL of thiodicarb and can be used in agriculture in both liquid fertilizers and direct application in irrigation or sprinkler systems.
- the culture medium that is used for B. megater ⁇ um is a nutritive medium, which consisted of peptone, distilled water, yeast extract and NaCl.
- stages were carried out, starting the processes with the conventional culture medium.
- stages El and E-2 Stage 1 and Stage
- stage 2 0.1 DL of thiodicarb (0.0000175 gr) is added to the culture medium maintaining a humidity of 32%, with mortalities between 70 to 68% (In stage 2 the same concentration of thiodicarb is used to reinforce the growth of resistant strains ).
- the resistant strains are transferred to solid culture media with 0.2 DL (0.000035 gr) of thiodicarb (E-3 and E-4, again in E-4 the same concentration of thiodicarb is used to reinforce the growth of resistant strains) presenting mortality of 65%.
- organisms At the end of stage E-4, organisms have been selected for 207 days.
- Stages E-5 and E-6 are subjected to a first addition of biphentrine; the culture medium is added with 0.1 DL of biphentrine (0.00000006 gr), presenting mortality of 66 to 63% (in the same way as the previous case, E-6 is used to reinforce the growth of resistant strains).
- Stages E-7 and E-8 are increased to 0.2 DL of biphentrine (0.00000012 gr) presenting mortality from 60 to 59%.
- E-8 step of strengthening the growth of resistant strains
- the adaptation process reaches 420 days.
- Stages E-9 and E-10 0.3 DL (0.000053 gr) of thiodicarb is added, presenting mortality of 55 to 54% (As in the previous stages, E-10 is for reinforcement of growth of resistant strains).
- stages E-ll and E-12 0.4 DL (0.000070 gr) of thiodicarb are added, with mortality of 53 to 51%.
- 618 days of selection process are reached.
- stages E-13 and E-14 0.3 DL (0.00000018 gr) of biphentrine is added, presenting mortality of 49 to 45% (As in the previous stages, E-14 is for reinforcement of growth of resistant strains).
- stages E-15 and E-16 it is increased to 0.4 DL (0.00000024 gr) presenting mortality of 45 to 40%.
- stage 16 step of strengthening the growth of resistant strains 794 days of selection process are reached.
- stages E-17 and E-18 0.5 DL (0.000087 gr) of thiodicarb is added, presenting mortality of 40 to 38% (As in the previous stages, E-18 is for reinforcement of growth of resistant strains).
- stage E-19 and E-20 0.6 DL (0.00011 gr) of thiodicarb is added, presenting mortality of 35 to 33%.
- E-21 and E-22 0.5 DL (0.00000030 gr) of biphentrine is added, presenting mortality of 35 to 33% (As in the previous stages, E-22 is for reinforcement of resistant strain growth) .
- E-22 is for reinforcement of resistant strain growth
- E-23 and E-24 it increases to 0.6 DL of biphentrine (0.00000036 gr) presenting mortality of 30 to 28%.
- E-24 step of strengthening the growth of resistant strains
- stages E-25 and E-26 0.7 DL (0.00012 gr) of thiodicarb is added, presenting mortality of 25 to 24% (As in the previous stages, E-26 is for reinforcement of growth of resistant strains).
- E-27 and E-28 0.8 DL (0.00014 gr) of thiodicarb are added, presenting mortality of 22 to 20%.
- E-28 step of strengthening the growth of resistant strains
- stages E-33 and E-34 the 0.9 DL (0.00016 gr) of thiodicarb, presenting mortality of 13 to 11% (As in the previous stages the E-34 is for reinforcement of growth of resistant strains).
- stages E-35 and E-36 1 DL (0.00018 gr) of thiodicarb is added, presenting mortality of 10 to 8%.
- E-36 step of strengthening the growth of resistant strains
- E-37 and E-38 add 0.9 DL of biphentrine (0.00000042 gr); presenting mortality of 8 to 6% (As in the previous stages the E-38 is for reinforcement of growth of resistant strains).
- the E-39 and E-40 is increased to 1 DL of biphentrine (0.00000048 gr) reducing mortality to 5%
- Bacillus subtillis It is a Gram positive, sporogenic, strict aerobic bacillus, with a thick layer of murein, its initial size is on average 0.75 mp ⁇ thick x 0.9 pm long; its ambient growth temperature ranges from 15 to 55 ° C, since the natural habitat of B. subtilis is the soil, which is subject to large temperature fluctuations.
- phenotypic change when the temperature is modified from 30 ° C to 45 ° C or 80 ° C. Its activity takes place within a pH of 4.7 to 5.5, a humidity of 70 to 80% and accepts levels of minimum toxic concentration, represented in traces.
- bacitracin an antibiotic which acts against negative Gram by damaging its cell membrane and inhibiting wall formation.
- enzymes such as bacterial amylase useful in the paper and textile industry, and enzyme applied to tan skins, remove stains and tenderize meat.
- the Microcosm Model was used (Kabir and cois, 1995). The strains were classified by traditional (numerical taxonomy) and immunochemical (indirect immunofluorescency-IFI, according to Llewot and Stead, 1991) methods. Pure strains from different sectors were used.
- the culture medium used for B. subtill ⁇ s is a nutritive medium, with 5% agar-soybean maintaining the pH parameters, minimum oxygen levels, humidity and temperature of its original state.
- stages were carried out, starting the processes with the conventional culture medium.
- stages El and E-2 Stage 1 and Stage 2
- 0.1 DL of thiodicarb (0.0000175 gr) is added to the culture medium.
- slow growth is recorded up to 72 hours.
- a mortality 82% and in the E-2 of 80%.
- Humidity and habitat temperature are maintained.
- stage 2 the same concentration of thiodicarb is used to reinforce the growth of resistant strains).
- the resistant strains are transferred to solid culture media with 0.2 DL (0.000035 gr) of thiodicarb (E-3 and E-4, again in E-4 the same concentration of thiodicarb is used for
- stage E-4 the organisms have been selected for 294 days.
- Stages E-5 and E-6 are subjected to a first addition of biphentrine; the culture medium is added with 0.1 DL of biphentrine (0.00000006 gr), presenting mortality of 72 to 71% (in the same way as the previous case, E-6 is used to reinforce the growth of resistant strains). Stages E-7 and E-8 are increased to 0.2 DL of biphentrine (0.00000012 gr) presenting mortalities between 70 to 68%. At the end of E-8 (step of strengthening the growth of resistant strains) the adaptation process reaches 570 days.
- Stages E-9 and E-10 0.3 DL (0.000053 gr) of thiodicarb is added, presenting mortality of 66 to 63% (As in the previous stages, E-10 is for reinforcement of growth of resistant strains).
- stages E-ll and E-12 0.4 DL (0.000070 gr) of thiodicarb are added, with a mortality rate of 64 to 62%.
- 827 days of selection process are reached.
- stages E-13 and E-14 0.3 DL (0.00000018 gr) of biphentrine is added, presenting mortality of 63 and in the next stage it increases to 65% (As in the previous stages the E-14 is for reinforcement of growth of resistant strains). In stages E-15 and E-16 it is increased to 0.4 DL (0.00000024 gr) presenting mortality of 64 to 60%. At the end of stage 16 (step of strengthening the growth of resistant strains) 1071 days of selection process are reached.
- stages E-17 and E-18 0.5 DL (0.000087 gr) of thiodicarb is added, presenting mortality of 59 to 54% (As in the previous stages the E-18 is for reinforcement of growth of resistant strains).
- stage E-19 and E-20 0.6 DL (0.00011 gr) of thiodicarb is added, presenting mortality of 52 to 50%.
- 1283 days of selection are counted.
- stages E-25 and E-26 0.7 DL (0.00012 gr) of thiodicarb is added, presenting mortality of 35 to 30% (As in the previous stages, E-26 is for reinforcement of growth of resistant strains).
- E-27 and E-28 0.8 DL (0.00014 gr) of thiodicarb are added, presenting mortality of 28 to 25%.
- E-28 step of strengthening the growth of resistant strains 1581 days of adaptation are reached.
- E-29 and E-30 0.7 DL of biphentrine (0.00000036 gr) is added, presenting mortality of 25 to 23% (As in the previous stages, E-30 is for reinforcement of growth of resistant strains).
- E-31 and E-32 it increases to 0.8 DL of biphentrine (0.00000042 gr), presenting mortality of 20 to 18%.
- E-32 step of strengthening the growth of resistant strains 1704 work days are reached.
- E-34 is for reinforcement of growth of resistant strains.
- E-35 and E-36 1 DL (0.00018 gr) of thiodicarb is added, presenting a 12 to 9% decrease in mortality.
- E-36 step of strengthening the growth of resistant strains
- E-37 and E-38 add 0.9 DL of biphentrine (0.00000042 gr); presenting mortality of 10 to 8% (As in the previous stages the E-38 is for reinforcement of growth of resistant strains).
- the E-39 and E-40 is increased to 1 DL of biphentrine (0.00000048 gr) maintaining the mortality of 6%
- Table No.3 Comparative Table Initial and Final Characteristics
- the microorganism exhibits a normal activity in said medium, phosphorus traces being found in its cell membrane (P 2 O 5 ). It is also observed that the organic material constituted in 80% of carbohydrates is stabilized in a period of 35 days on average, unlike the wild microorganism with which the time was 120 days.
- Cellular membrane is completely smooth and its activity develops within a pH of 6.5 to 7.2 with a humidity of 60%.
- B. thuringiensis cells form crystalline inclusions visible under the optical microscope during sporulation. These crystalline inclusions are composed of protoxins, which contain d-endotoxins, of specific insecticidal activity against insects.
- Some varieties of Bacillus thuringiensis can also produce another toxin, b-exotoxin. It is a toxin that is produced during vegetative growth, and is a nucleotide derivative of adenine that functions as an RNA polymerase inhibitor. But the use of this toxin is prohibited in some countries, as it is also toxic to mammals.
- the culture medium to maintain B. thuringiensis is a nutritive medium, with 5% agar-soybean maintaining the parameters of pH, minimum levels of oxygen, humidity and temperature of its original state.
- stages El and E-2 Stage 1 and Stage 2
- 0.1 DL of thiodicarb 0.0000175 gr
- Humidity and habitat temperature are maintained.
- stage 2 the same concentration of thiodicarb is used to reinforce the growth of resistant strains).
- the resistant strains are transferred to solid culture media with 0.2 DL (0.000035 gr) of thiodicarb (E-3 and E-4, again in E-4 the same concentration of thiodicarb is used to reinforce the growth of resistant strains)
- stage E-4 the organisms have been selected for 282 days.
- Stages E-5 and E-6 are subjected to a first addition of biphentrine; the culture medium is added with 0.1 DL of biphentrine (0.00000006 gr), presenting mortality of 68 to 65% (in the same way as the previous case, E-6 is used to reinforce the growth of resistant strains). Stages E-7 and E-8 are increased to 0.2 DL of biphentrine (0.00000012 gr) presenting mortality from 64 to 63%. At the end of E-8 (step of strengthening the growth of resistant strains) the adaptation process reaches 565 days.
- Stages E-9 and E-10 0.3 DL (0.000053 gr) of thiodicarb is added, presenting mortality of 65 to 61% (As in the previous stages the E-10 is for reinforcement of growth of resistant strains). In stages E-ll and E-12, 0.4 DL (0.000070 gr) of thiodicarb are added, with mortality of 57 to 55%. At the end of stage E-12 (step of strengthening the growth of resistant strains), 822 days of selection process are reached.
- stages E-13 and E-14 0.3 DL (0.00000018 gr) of biphentrine is added, with mortality of 57 to 55% (As in the previous stages, E-14 is for the reinforcement of growth of resistant strains).
- stages E-15 and E-16 it is increased to 0.4 DL (0.00000024 gr) presenting mortality from 52 to 50%.
- stage 16 step of strengthening the growth of resistant strains 1064 days of selection process are reached.
- stages E-17 and E-18 0.5 DL (0.000087 gr) of thiodicarb is added, presenting mortality of 52 to 50% (As in the previous stages, E-18 is for the reinforcement of growth of resistant strains).
- stage E-19 and E-20 0.6 DL (0.00011 gr) of thiodicarb is added, presenting mortality of 50 to 45%.
- 1280 days of selection are counted.
- E-21 and E-22 0.5 DL (0.00000030 gr) of biphentrine is added, presenting mortality of 50 to 45% (As in the previous stages, E-22 is for reinforcement of growth of resistant strains) .
- E-23 and E-24 is increased to 0.6 DL of biphentrine (0.00000036 gr) presenting mortality from 44 to 42%.
- stages E-25 and E-26 0.7 DL (0.00012 gr) of thiodicarb is added, presenting mortality of 44 to 40% (As in the previous stages, E-26 is for reinforcement of growth of resistant strains).
- E-27 and E-28 0.8 DL (0.00014 gr) of thiodicarb are added, presenting mortality of 36 to 35%.
- E-28 step of strengthening the growth of resistant strains
- E-29 and E-30 For E-29 and E-30, 0.7 DL of biphentrine (0.00000036 gr) is added, presenting mortality of 36 to 33% (As in the previous stages, E-30 is for the reinforcement of growth of resistant strains). In E-31 and E-32, it increases to 0.8 DL of biphentrine (0.00000042 gr) presenting mortality of 30 to 28%. At the end of E-32 (step of strengthening the growth of resistant strains) 1777 work days are reached.
- stages E-33 and E-34 the 0.9 DL (0.00016 gr) of thiodicarb, presenting 30% mortality (As in the previous stages the E-34 is for reinforcement of
- E-37 and E-38 add 0.9 DL of biphentrine (0.00000042 gr); presenting mortality of 30 to 28% (As in the previous stages the E-38 is for reinforcement of growth of resistant strains).
- the E-39 and E-40 is increased to 1 DL of biphentrine (0.00000048 gr) maintaining mortality at 18 to 17% At the end of the 40 stage we have 2015 working days.
- stages E-41 and E-42 the 1.1 DL (0.00020 gr) of thiodicarb, presenting mortality of 18 to 17% (As in the previous stages the E-42 is for reinforcement of growth of resistant strains).
- stages E-43 and E-44 1.2 DL (0.00022 gr) of thiodicarb are added, presenting mortality in the 2 stages of 14%.
- stages E-45 and E-46 the 1.1 DL (0.00000054 gr) of biphentrine, presenting mortality of 15 to 12% (As in the previous stages, the E-46 is for reinforcement of growth of resistant strains).
- stages E-47 and E-48 1.2 DL (0.00000060 gr) of Biofentrine is added, presenting mortality of 10 to 7%.
- Clostridium pasteuranium It is a Gram positive, anaerobic and heterotrophic bacillus, its natural habitat is the soil, it has a mechanism for phosphorylation and electron transport, so that ATP obtains it through phosphorylation at the substrate level. Its optimal development is presented in a temperature range of 20 to 30 ° C. They are mobile, they move using a scourge, it is chemoorganotrophic. This bacillus is facultative anaerobic.
- stages E-1 and E-2 Stage 1 and Stage 2
- 0.1 DL of thiodicarb 0.0000175 gr
- Humidity and habitat temperature are maintained.
- stage 2 the same concentration of thiodicarb is used to reinforce the growth of resistant strains).
- the resistant strains are transferred to solid culture media with 0.2 DL (0.000035 gr) of thiodicarb (E-3 and E-4, again in E-4 the same concentration of thiodicarb is used to reinforce the growth of resistant strains) presenting mortality of 83 to 80%.
- E-4 thiodicarb
- Stages E-5 and E-6 are subjected to a first addition of biphentrine; the culture medium is added with 0.1 DL of biphentrine (0.00000006 gr), presenting mortality of 81
- Stages E-9 and E-10 0.3 DL (0.000053 gr) of thiodicarb is added, presenting mortality of 70 to 69% (As in the previous stages, E-10 is for the reinforcement of growth of resistant strains).
- Stage E-ll and E-12 0.4 DL (0.000070 gr) of thiodicarb is added, with mortalities of 60 to 67%.
- 803 days of selection process 803 days of selection process are reached.
- stages E-13 and E-14 0.3 DL (0.00000018 gr) of biphentrine is added, presenting mortality of 66 to 63% (As in the previous stages, E-14 is for reinforcement of growth of resistant strains). In stages E-15 and E-16 it is increased to 0.4 DL (0.00000024 gr), presenting mortalities between 60 and 58%. At the end of the
- stage 16 (step of strengthening the growth of resistant strains) 1027 days of selection process are reached.
- stages E-17 and E-18 0.5 DL (0.000087 gr) of thiodicarb is added, presenting mortality of 58 to 55% (As in the previous stages, E-18 is for growth reinforcement of resistant strains).
- stage E-19 and E-20 0.6 DL (0.00011 gr) of thiodicarb is added, with mortalities of 55 to 51%.
- 1211 days of selection are counted.
- E-21 and E-22 0.5 DL (0.00000030 gr) of biphentrine is added, presenting mortality of 48 to 46% (As in the previous stages, E-22 is for reinforcement of resistant strain growth) .
- E-23 and E-24 it is increased to 0.6 DL of biphentrine (0.00000036 gr), presenting mortalities between 40 and 38%.
- E-24 step of strengthening the growth of resistant strains
- E-29 and E-30 For E-29 and E-30, 0.7 DL of biphentrine (0.00000036 gr) is added, presenting mortality of 29 to 25% (As in the previous stages, E-30 is for reinforcement of growth of resistant strains). In the E-31 and E-32 it is increased to 0.8 DL of biphentrine (0.00000042 gr) presenting mortalities between 22 to 18%. At the end of E-32 (step of strengthening the growth of resistant strains) 1631 work days are reached.
- stages E-33 and E-34 the 0.9 DL (0.00016 gr) of thiodicarb, presenting mortality of 18 to 16% (As in the previous stages the E-34 is for reinforcement of growth of resistant strains).
- stages E-35 and E-36 1 DL (0.00018 gr) of thiodicarb is added, presenting mortality of 13 to 12%.
- E-36 step of strengthening the growth of resistant strains
- E-37 and E-38 add 0.9 DL of biphentrine (0.00000042 gr); presenting mortality of 10% in the two stages, (As in the previous stages the E-38 is for reinforcement of growth of resistant strains).
- the E-39 and E-40 is increased to 1 DL of biphentrine (0.00000048 gr) maintaining mortality at 8 to 5% At the end of the 40 stage we have 1777 work days.
- stage 2 the same concentration of thiodicarb is used to reinforce the growth of resistant strains).
- the resistant strains are transferred to solid culture media with 0.2 DL (0.000035 gr) of thiodicarb (E-3 and E-4, again in E-4 the same concentration of thiodicarb is used to reinforce the growth of resistant strains) presenting 60% mortality in both stages.
- stage E-4 the organisms have been selected for 253 days.
- Stages E-5 and E-6 are subjected to a first addition of biphentrine; the culture medium is added with 0.1 DL of biphentrine (0.00000006 gr), presenting mortality of 62 to 60% (in the same way as the previous case, E-6 is used to reinforce the growth of resistant strains). Stages E-7 and E-8 are increased to 0.2 DL of biphentrine (0.00000012 gr) presenting mortality of 58 to
- Stages E-9 and E-10 0.3 DL (0.000053 gr) of thiodicarb is added, presenting mortality of 55 to 53% (As in the previous stages, E-10 is for reinforcement of growth of resistant strains).
- stages E-ll and E-12 0.4 DL (0.000070 gr) of thiodicarb are added, with mortality of 51 to 49%.
- stage E-12 step of strengthening the growth of resistant strains 727 days of selection process are reached.
- stages E-13 and E-14 0.3 DL (0.00000018 gr) of biphentrine is added, presenting mortality of 49 to 47% (As in the previous stages, E-14 is for reinforcement of growth of resistant strains).
- stages E-15 and E-16 it is increased to 0.4 DL (0.00000024 gr) presenting mortality from 44 to 42%.
- stage 16 step of strengthening the growth of resistant strains 936 days of selection process are reached.
- stages E-17 and E-18 0.5 DL (0.000087 gr) of thiodicarb is added, presenting mortality of 41 to 40% (As in the previous stages the E-18 is for reinforcement of
- E-21 and E-22 0.5 DL (0.00000030 gr) of biphentrine is added, presenting mortality of 36 to 35% (As in the previous stages, E-22 is for reinforcement of resistant strain growth) .
- E-23 and E-24 it is increased to 0.6 DL of biphentrine (G.00000036 gr) presenting mortality of 33 to 31%.
- E-24 step of strengthening the growth of resistant strains
- stages E-25 and E-26 0.7 DL (0.00012 gr) of thiodicarb is added, presenting mortality of 30 to 28% (As in the previous stages, E-26 is for the reinforcement of growth of resistant strains).
- E-27 and E-28 0.8 DL (0.00014 gr) of thiodicarb are added, with mortality of 25 to 23%.
- E-28 step of strengthening the growth of resistant strains
- E-29 and E-30 For E-29 and E-30, 0.7 DL of biphentrine (0.00000036 gr) is added, presenting mortality of 21 to 18% (As in the previous stages, E-30 is for reinforcement of growth of resistant strains). In E-31 and E-32, it increases to 0.8 DL of biphentrine (0.00000042 gr) presenting mortality of 15 to 14%. At the end of E-32 (step of strengthening the growth of resistant strains) 1505 work days are reached.
- stages E-33 and E-34 the 0.9 DL (0.00016 gr) of thiodicarb, presenting mortality of 25 to 22% (As in the previous stages the E-34 is for reinforcement of growth of resistant strains).
- stages E-35 and E-36 1 DL (0.00018 gr) of thiodicarb is added, presenting mortality of 20 to 18%.
- At the end of the E-36 we have 1601 days of adaptation.
- E-37 and E-38 add 0.9 DL of biphentrine (0.00000042 gr); presenting mortality of 16 to 14% (As in the previous stages the E-38 is for reinforcement of growth of resistant strains).
- the E-39 and E-40 is increased to 1 DL of biphentrine (0.00000048 gr) maintaining the
- the microorganism subjected to poisoning with traces of thiodicarb and biphentrine has normal activity in said medium, with traces of Nitrogen in its cell membrane.
- the purity of the strain is checked, by taking a 0.1 ml aliquot of the microorganism culture suspension and inoculating a Triptona Soya Agar plate (TSA) which is incubated at 52 ° C +/- 2 ° C, its mobility is checked by staining with malachite green Gram.
- TSA Triptona Soya Agar plate
- a suspension of this microorganism is carried out in a TSA plate that is incubated from 48 to 72 h at 52 ° C. +/- 2 ° C, subsequently, with a sterile swab, a sample is taken and 100 ml of liquid medium containing 0.01 DL of biphentrine and / or 0.01 DL of thiodicarb are inoculated, placed under stirring at 37 ° C and 170 rpm for 4 hours; subsequently the biomass is resuspended in 50 ml of
- the medium containing different lioprotective solutions used to prepare microorganisms for lyophilization
- the medium is homogenized and the ÜFC / mi count is determined.
- This inoculum is taken to the incubator and periodically reseeded in the same culture medium. This procedure is performed in periods of time ranging from 72 hours to 120 hours; All these procedures are performed periodically to date. Maintenance time under the same conditions is used to make general observations.
- strains are kept in active culture medium, adding a previously sterilized mineral oil, in this way the required humidity is guaranteed.
- Patent No. 26662 granted by the
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US16/619,181 US11470848B2 (en) | 2018-02-19 | 2019-01-30 | Consortium of (carbamate) thiodicarb-resistant and (pyrethroid) biphenthrin-resistant bacteria and use thereof in liquid fertilizers |
EP19754042.0A EP3732970A4 (en) | 2018-02-19 | 2019-01-30 | CONSORTIUM OF (CARBAMATE) THIODICARB RESISTANT AND (PYRETHROID) BIPHENTHRIN RESISTANT BACTERIA AND THEIR USE IN LIQUID FERTILIZERS |
BR112020019693A BR112020019693A8 (pt) | 2018-02-19 | 2019-01-30 | Consórcio de bactérias resistentes ao tiodicarbe (carbamato) e bifentrina (pirretroide) e sua utilização em fertilizantes líquidos |
CR20200426A CR20200426A (es) | 2018-02-19 | 2019-01-30 | Consorcio de hongos resistentes a tiocarb (carbamato) y bifentrina (piretroide) y su uso en abonos líquidos |
DO2020000193A DOP2020000193A (es) | 2018-02-19 | 2020-10-26 | Consorcio de bacterias resistentes a tiodicarb (carbamato) y a bifentrina (piretroide) y su uso en abonos líquidos. |
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MX2018002063A MX2018002063A (es) | 2018-02-19 | 2018-02-19 | Proceso para hacer resistente a tiodicarb (carbamato) y bifentrina (piretroide) a un consorcio de bacterias nitrificantes, solubilizadores de fosforo y antagonistas de algunos patogenos, para ser usados en bioabonos liquidos para ser aplicados de manera edafica y/o foliar. |
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WO2006003777A1 (ja) | 2004-07-05 | 2006-01-12 | Kazuo Okano | イオンバランスセンサ |
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MX347762B (es) * | 2006-04-04 | 2017-05-11 | Orlando Castro Cabrera Luis | Compuesto de microorganismos adaptados para biodegradar la fraccion organica contenida en residuos solidos y proceso para su preparacion. |
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US8569028B2 (en) | 2010-06-09 | 2013-10-29 | The United States of America, as represented by the Secretary of the Agriculture | Method for encapsulation of microparticles |
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MX347762B (es) * | 2006-04-04 | 2017-05-11 | Orlando Castro Cabrera Luis | Compuesto de microorganismos adaptados para biodegradar la fraccion organica contenida en residuos solidos y proceso para su preparacion. |
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