(19)
(11)EP 3 732 970 A1

(12)EUROPEAN PATENT APPLICATION
published in accordance with Art. 153(4) EPC

(43)Date of publication:
04.11.2020 Bulletin 2020/45

(21)Application number: 19754042.0

(22)Date of filing:  30.01.2019
(51)International Patent Classification (IPC): 
A01N 63/02(2006.01)
C12N 1/20(2006.01)
C05F 11/08(2006.01)
(86)International application number:
PCT/MX2019/000005
(87)International publication number:
WO 2019/160399 (22.08.2019 Gazette  2019/34)
(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
Designated Extension States:
BA ME
Designated Validation States:
KH MA MD TN

(30)Priority: 19.02.2018 MX 2018002063

(71)Applicant: Salus Mundi Investments Limited
Ciudad de México, 06600 (MX)

(72)Inventor:
  • CASTRO CABRERA, Luis Orlando
    Ciudad de México, 06600 (MX)

(74)Representative: Gallego Jiménez, José Fernando 
Ingenias Creaciones, Signos e Invenciones S.L.P. Avda. Diagonal 514, 1º 4ª
08006 Barcelona
08006 Barcelona (ES)

  


(54)CONSORTIUM OF (CARBAMATE) THIODICARB-RESISTANT AND (PYRETHROID) BIPHENTHRIN-RESISTANT BACTERIA AND USE THEREOF IN LIQUID FERTILISERS


(57) The consortium comprises the Clostridium pasteuranium, Bacillis thuringiensis, Bacillus megaterium, Bacillus subtilis, Azotobacter vinelandii and Rhizobium sp micro-organisms, which are subjected to increasing concentrations of thiodicarb and biphenthrin in order to render them tolerant to these compounds without manipulating their genome. The use of the consortium in the form of a liquid biofertiliser enriches the soil with bacteria which, besides being resistant to thiodicarb and biphenthrin, capture atmospheric nitrogen, solubilise phosphor and act against some pathogens.


Description

Invention background



[0001] Since the TECHNIQUE STATUS regarding the scientific investigation and application of technology targeted to induce growth, repopulation and development of a consortium of beneficial microorganisms that can tolerate trace chemicals on soils and organic residues, and that are resistant to new generations of substances replacing conventional agrochemicals (forbidden in agriculture), is stated mainly in PATENT No.PA/2006/003777, issued by the IMPI (April 27, 2017) where consortia of microorganisms resistant to organochlorinated and organophosphate compounds were isolated, in this new request we describe the intent to develop an INVENTIVE ACTIVITY to develop, grow and select consortia of aerobic microorganisms resistant to chemical compounds in new generations of pesticides and biological control such as pyrethroid and carbamate compounds that are supposed to have a controlled use because of their secondary effects that pollute the soils, water mantles, and effect on agricultural development. In this document, the INNOVATION is formed by developing a consortium of specific bacterial species, adapted to resist, tolerate and mineralize the organic matter polluted with carbamate such as thiodicarb and pyrethroids such as bifenthrin, these compounds are currently used in the agricultural and livestock industries, and are present in the organic residues. Liquid Biofertilizers are produced with this organic residues (for example, horse manure, sugar cane waste, dairy, pancreatic enzymes, carbohydrates, etc.); this microbial consortium enriches them and a produce with a bacterial micro load is obtained, when applied to crops, capture atmospheric nitrogen and make it available to the plants, phosphorous available in soils is dissolved as P2O4 by adding one O and turning it into P2O5 available to be used by plants, it contains microorganisms antagonist to some pathogens and repopulates the soil with beneficial bacteria. It is worth mentioning that the microorganisms forming our consortia ARE NOT GENETICALLY MODIFIED nor are human, animal or plant pathogens.

[0002] With the use of organochlorinated and organophosphate compounds banned as pesticides on the second half of the twentieth century, carbamate and pyrethroid compounds become an alternative. Carbamate are pesticide substances formed by a N atom bonded to a labile group, the carbamic acid; its main characteristic is its high toxicity, low chemical stability and null build up on tissues; and pyrethroids are molecules with pesticide activity, they remain longer in the environment because the chemical modification of their formula makes them more stable under sunlight and to heat. These products are used indiscriminately in the agricultural and livestock industries, generating residues.

[0003] This research is outlined within the guidelines of the United Nation's Agenda 21 (Chapters 10, 11, 12 and 14, accordingly), regarding desertification and drought; agriculture and sustainable development. Calculations show that soil degradation on a world scale extends over 2000 million of hectares, endangering the way of life for over 1000 million people. Calculations show that approximately 2/5 of Earth's surface is dry land, with a limited fresh water supply, and a high percentage of these are eroded. Approximately 65% of the cultivable land has already lost a physical or a biological function.

[0004] In Latin America, indiscriminate use of agrochemicals for decades in agriculture has left residues, polluting the soil, surface and underground water. "Pesticides are designed to kill, reduce and repel insects, weeds, rodents, mushrooms and other organisms that could harm public health and the nation's economies. When these chemical products are handled or disposed improperly, they can harm human health". "The main risks linked to human health for being exposed chronically to small doses are related with cancer, birth defects, nervous and endocrine system disorders".

[0005] Statements from: Childhood Pesticides Poisoning: Information for Advocacy and Action", UNEP Chemicals, May 2004.

[0006] Developing our scientific research, we have found soils with absence of micro loads caused by the excessive use of agrochemicals used on them. Example: Rice growing in the lbaqué area (Colombia). Tomato growing in Sinaloa (Mexico). Soy growing in the Santa Fe province (Argentina, among other cases).

[0007] The environmental awareness, ecological knowledge, attitudes and values towards the environment have been growing within our communities. The problem with municipal, home, agricultural and livestock solid residues keeps growing and its production is excessive, the lack of separation from the source, incorrect disposal, lack of areas to handle them and lack of treatment or recovery. Among the main problems caused by the production of gases that pollute the atmosphere, leachates that pollute the soils, underground water, surface water and generation of sources for disease or vehicles for disease transmission. The current solid organic residues from any source are very different to the ones produced 20 years ago related to the accumulation of trace chemicals, this toxicity is directly related to the pesticide, herbicide and acaricide evolution, among others.

[0008] International organizations, such as the FAO (Food and Agriculture Organization) and the WHO (World Health Organization), have established the maximum allowed levels of pesticide ingestion, however, national authorities in each country are responsible for establishing the proper legislation and carefully monitoring its use and the amount of residue through adequate analytical controls.

[0009] This is a scientific research with aerobic, phosphorus solubilizing bacteria, antagonist to pathogens from Dr. Luis Orlando Castro Cabrera's strain base, it is worth mentioning that the microorganisms used within this research were isolated originally by Dr. Luis Orlando Castro Cabrera's group according to patent 11851 from Colombia, such strains have been worked with since 1984.

[0010] The strains have been submitted to several stress stages using induced changes, adding traces to obtain chemical resistance to the synthetic pyrethroid bifenthrin (2- methyl biphenyl -3-ilmetil(2)-(1RS,3RS)-3-(2-chlorine 3,3,3- trifluoro propene)-2,2-dimethyl-cyclo-propane-carboxylate; alphanumeric code CA DPR Chem Code 2300. CAS 82657-04-3. CIPAC 415. FMC 54800. PC Code 128825) and the thiodicarb carbamate (C10H18N4O4S3).

[0011] It is worth mentioning that the microorganism consortia here described have been deposited before the INIFAP at the National Center of Genetic Resources under accession number by the INTERNATIONAL DEPOSIT AUTHORITY: CM-CNRG TB46 (Deposit certificates are annexed). Using these consortia for the production of liquid fertilizers, we are supplying a safe and effective product for agricultural use to control pests as an alternative to chemical insecticides and pesticides.

[0012] The following isolation protocol for resistant strains and the formation of the microbial consortium shows that every one of the strains was evolved towards attained resistance WITHOUT GENOME MANIPULATION for each organism. On the other hand, each strain was isolated and selected independently through consecutive strain growth increasing the synthetic pyrethroid concentrations, such as bifenthrin and carbamates such as thiodicarb.

[0013] Percentages for each microorganism belonging to the consortium vary from 20 to 30% of the CFU for each of the strains. Amount variability will depend of the soil characteristics for the consortium.

Procedure description



[0014] To develop each of the research stages, logistics are defined with the two (2) groups of ingredients (thiodicarb and bifenthrin) as follows:

Materials and methods



[0015] Bacteria strains, nitrifiers, phosphorus solubilizers and antagonists for pathogens:
  1. a) Azotobacter vinelandii
  2. b) Bacillus megaterium
  3. c) Bacillus subtillis
  4. d) Bacillus thuringiensis
  5. e) Clostridium pasteuranium
  6. f) Rhizobium

Preparation for stage development



[0016] 
  1. 1. Glass material is sterilized in an autoclave.
  2. 2. The required amount of growth media is prepared, DFA Agar, Malt Agar (Corn Agar) and (PDA) to obtain viable colonies.
  3. 3. Lethal dose (LD) applied to a hectare is established, for thiodicarb as well as bifenthrin. 10 Petri dishes are prepared with 0.1 LD of thiodicarb of bifenthrin. Dishes are incubated at 25ºC for 72 hours with cold light.

Methodology



[0017] 
  1. a. Petri dishes are placed inside the laminar flow chamber along with the diluted samples, to seed microorganisms in the dishes, a one ml sample of the strain is taken with an automated pipette, 10-1 and 10-2 solutions are performed and each solution is homogenized.
  2. b. Having the petri dishes with the sterile and cold growth media, it is homogenized with 6 right to left movements, 6 clockwise movements, 6 counterclockwise movements and 6 front to back movements over a flat horizontal surface until the inoculum is completely integrated into the media, stand until it solidifies and each dish is covered with parafilm to avoid contamination during incubation.
  3. c. Dishes with thiodicarb and Bifenthrin are placed inside the incubator at 25ºC for 72 hours with a cold light lamp.
  4. d. Inoculated dishes are incubated for 72 hours and the bacteria growth will be recorded, the product concentration impact is determined over the plate growth (12 hours of incubation).
  5. e. The residual impact of the product is reviewed after 7 days in the malt agar and PDA agar medias.
  6. f. If there is no growth over 10%, the process is repeated until a mortality under 90% is obtained.
  7. g. The colony diameter is determined using a graduated ruler, data is recorded subtracting the diameter of the seeded dish, from which the effect over the mycelial growth will be determined compared to the control (not poisoned MA, denominated as percentage of inhibition of radial growth (PIRG %).
  8. h. Progress is recorded daily and after 7 days analysis of an acceptable coverage is performed, if not, the procedure is repeated as many times as required.
  9. i. From the 10 Petri dishes for each microorganism, the one with the lowest mortality is the one that will continue with the following study.
  10. j. The study is performed again using this percentage until coverage increases and so on, the addition of thiodicarb and bifenthrin is alternated with the LD in intervals of 0.1 LD; all these experiments have been designed with the experimental strains and a control.
  11. k. With the data obtained from the bacterial growth, the percentage of inhibition of gradual growth is obtained, the data is transformed for its analysis with the expression 2 arc sin. A two-way classification analysis of variance is applied and later the Turkey test is performed using a 5% probability.

Records



[0018] Characteristics for this procedure were recorded during all stages through the following indicators:
  1. a. Growth speed
  2. b. Percentage of inhibition measured in Number of CFU/mm
  3. c. Percentage of poison resistance

Stages performed on each microorganism



[0019] For each species, stages analyzing times required to cover the entire field were performed. Once this time was determined, they were left to rest for 10 days to be later subject to the following protocol:

Azotobacter vinelandii



[0020] It is a Gram-negative bacillus, facultative aerobe, grown in a low oxygen concentration, its natural habitat is soil, swamp soil and water; they are chemoorganotrophs. They have a complex cellular wall, formed by an external membrane and an internal peptidoglycan layer containing muramic acid and murein, making it a N fixing bacteria, capturing it from the atmosphere and storing it in its murein layer. The optimal development is seen in a temperature between 30 to 37ºC.

Stages performed in the lab using Azotobacter vinelandii



[0021] Beginning with habitat conditions Humidity: 90% - Temperature: 30 to 37 ºC. pH: 7.0 to 7.5. These conditions are the starting point to adapt the strain to the new pollutants: thiodicarb and bifenthrin.

[0022] To adapt this microorganism, 40 stages were performed starting the processes with the conventional growth media. During stages E-1 and E-2 (Stage 1 and Stage 2) 0.1 LD (0.0000175 gr) of thiodicarb are added to the growth media. After incubation, a slow growth is recorded for 72 hours, until incubation day 45 with a mortality from 70 to 72%. The remaining 28% is under daily observation for 10 days to continue with the adaptation of this microorganism. Habitat humidity and temperature are maintained. (On stage 2, the same concentration of thiodicarb is used to strengthen the growth of resistant strains). The resistant strains are transferred to solid growth media with 0.2 LD (0.000035 gr) of thiodicarb (E-3 and E-4, again on E-4 the same concentration of thiodicarb is used to strengthen the growth of resistant strains) with a mortality between 66 to 62%. At the end of stage E-4, organisms have been selected for 164 days.

[0023] Stages E-5 and E-6 are submitted to the first addition of bifenthrin; 0.1 LD (0.00000006 gr) of bifenthrin are added, with a mortality from 62 to 61% (in the same way as the previous case, E-6 is used to strengthen the growth of resistant strains). On stages E-7 and E-8 bifenthrin is increased to 0.2 LD (0.00000012 gr) with a 60% mortality. At the end of E-8 (growth strengthening stage for the resistant strains) the adaptation process reaches 359 days.

[0024] On stages E-9 and E-10, 0.3 LD (0.000053 gr) of thiodicarb are added, with a mortality from 55 to 54%. (The same way as in previous stages, E-10 is used to strengthen the growth of resistant strains). On stages E-11 and E-12 0.4 LD (0.000070 gr) of thiodicarb are added, with a mortality rate from 52 to 48%. At the end of stage E-12 (growth strengthening stage for the resistant strains) the selection process reaches 525 days.

[0025] On stages E-13 and E-14, 0.3 LD (0.00000018 gr) of bifenthrin are added, with a mortality from 52 to 48% (The same way as in previous stages, E-14 is used to strengthen the growth of resistant strains). On stages E-15 and E-16, thiodicarb is increased to 0.4 LD (0.00000024 gr) with a 45% mortality. At the end of stage 16 (growth strengthening stage for the resistant strains) the selection process reaches 678 days.

[0026] On stages E-17 and E-18, 0.5 LD (0.000087 gr) of thiodicarb are added, with a mortality from 42 to 40% (The same way as in previous stages, E-18 is used to strengthen the growth of resistant strains). On stages E-19 and E-20, 0.6 LD (0.00011 gr) of thiodicarb are added, with a mortality rate from 35 to 32%. At the end of stage E-20 (growth strengthening stage for the resistant strains) the selection process reaches 816 days.

[0027] On stages E-21 and E-22, 0.5 LD (0.00000030 gr) of bifenthrin are added, with a mortality from 30 to 28% (The same way as in previous stages, E-22 is used to strengthen the growth of resistant strains). On stages E-23 and E-24, thiodicarb is increased to 0.6 LD (0.00000036 gr) with a mortality rate from 25 to 23%. At the end of stage 24 (growth strengthening stage for the resistant strains) the selection process reaches 934 days.

[0028] On stages E-25 and E-26, 0.7 LD (0.00012 gr) of thiodicarb are added, with a mortality from 25 to 22% (The same way as in previous stages, E-26 is used to strengthen the growth of resistant strains). On stages E-27 and E-28, 0.8 LD (0.00014 gr) of thiodicarb are added, with a mortality rate from 20 to 18%. At the end of stage E-28 (growth strengthening stage for the resistant strains) the selection process reaches 1039 days.

[0029] On stages E-29 and E-30, 0.7 LD (0.00000036 gr) of bifenthrin are added, with a mortality from 18 to 16% (The same way as in previous stages, E-30 is used to strengthen the growth of resistant strains). On stages E-31 and E-32, thiodicarb is increased to 0.8 LD (0.00000042 gr) with a mortality rate from 13 to 10%. At the end of stage 232 (growth strengthening stage for the resistant strains) the selection process reaches 1131 days.

[0030] On stages E-33 and E-34, 0.9 LD (0.00016 gr) of thiodicarb are added, with a mortality of 10% (The same way as in previous stages, E-34 is used to strengthen the growth of resistant strains). On stages E-35 and E-36, 1 LD (0.00018 gr) of thiodicarb are added, with a mortality rate from 35 to 32%. At the end of stage E-36 (growth strengthening stage for the resistant strains) the selection process reaches 1208 days.

[0031] On stages E-37 and E-38, 0.9 LD (0.00000042 gr) of bifenthrin are added, with a mortality from 10% (The same way as in previous stages, E-38 is used to strengthen the growth of resistant strains). On stages E-39 and E-40, thiodicarb is increased to 1 LD (0.00000048 gr) with a mortality rate from 8 to 7%. At the end of stage 40 (growth strengthening stage for the resistant strains) the selection process reaches 1278 days.

[0032] On this stage, the microorganism is already resistant to traces of 1 LD of bifenthrin and 1 LD of thiodicarb and can be used in agriculture in liquid fertilizers as well as applied directly on sprinkling irrigation systems.
Table No. 1. Comparative Table, Initial and Final Characteristics
SELECTIVE PROCESS CHARACTERISTICS A. Vinelandii
PARAMETERINITIALFINAL
Ph 6.5 - 7.2 7.5
Humidity 10% 100%
Respiration Aerobic Aerobic
Temperature 30 - 37°C 25ºC
Size 1.0 x 3.0 µm 1.0 x 3.0 µm
Resistant to: Organochlorinated, organic phosphorus and mercury compounds 1 LD of thiodicarb and bifenthrin

Bacillus meaaterium



[0033] It is a Gram-positive bacillus, spore forming, aerobic or facultative anaerobic bacteria; its initial average size is 1.5 µm wide by 5 µm long, its optimal growth temperature goes from 3 to 45ºC and develops within a pH from 3 to 5.5, with 60% humidity. Spores produced by these bacteria allow it to support hostile environments, either heat or drought. The growth media used for the B. megaterium is a nutritive media based in peptone, distilled water, yeast extract and NaCl.

Stages performed in the lab using bacillus megaterium



[0034] This scientific research began with habitat conditions obtained for the adaptation of the Bacillus megaterium to organophosphate compounds, organochlorinated and mercury for the microorganism at the E-43 pH: 7.2, Humidity: 28% - Temperature: Thermoresistant. Aerobic. These conditions are the starting point to adapt the strain to the new pollutants: thiodicarb (carbamate) and bifenthrin (pyrethroid). During all stages, we will be using a cold light lamp to adapt the microorganism to sunlight.

[0035] To adapt this microorganism, 40 stages were performed, beginning the process with the conventional growth media. During stages E-1 and E-2 (Stage 1 and Stage 2) 0.1 LD (0.0000175 gr) of thiodicarb are added maintaining 32% humidity, with mortality from 70 to 68% (On stage 2, the same concentration of thiodicarb is used to strengthen the growth of resistant strains). The resistant strains are transferred to solid growth media with 0.2 LD (0.000035 gr) of thiodicarb (E-3 and E-4, again on E-4 the same concentration of thiodicarb is used to strengthen the growth of resistant strains) with a 65% mortality rate. At the end of stage E-4, organisms have been selected for 207 days.

[0036] Stages E-5 and E-6 are submitted to the first addition of bifenthrin; 0.1 LD (0.00000006 gr) of bifenthrin are added, with a 63% mortality rate (in the same way as the previous case, E-6 is used to strengthen the growth of resistant strains). On stages E-7 and E-8 bifenthrin is increased to 0.2 LD (0.00000012 gr) with a mortality from 60 to 58%. At the end of E-8 (growth strengthening stage for the resistant strains) the adaptation process reaches 420 days.

[0037] On stages E-9 and E-10, 0.3 LD (0.000053 gr) of thiodicarb are added, with a mortality from 55 to 54%. (The same way as in previous stages, E-10 is used to strengthen the growth of resistant strains). On stages E-11 and E-12 0.4 LD (0.000070 gr) of thiodicarb are added, with a mortality rate from 53 to 51%. At the end of stage E-12 (growth strengthening stage for the resistant strains) the selection process reaches 618 days.

[0038] On stages E-13 and E-14, 0.3 LD (0.00000018 gr) of bifenthrin are added, with a mortality from 49 to 45% (The same way as in previous stages, E-14 is used to strengthen the growth of resistant strains). On stages E-15 and E-16, thiodicarb is increased to 0.4 LD (0.00000024 gr) with a mortality from 45 to 40%. At the end of stage 16 (growth strengthening stage for the resistant strains) the selection process reaches 794 days.

[0039] On stages E-17 and E-18, 0.5 LD (0.000087 gr) of thiodicarb are added, with a mortality from 40 to 38% (The same way as in previous stages, E-18 is used to strengthen the growth of resistant strains). On stages E-19 and E-20, 0.6 LD (0.00011 gr) of thiodicarb are added, with a mortality rate from 35 to 33%. At the end of stage E-20 (growth strengthening stage for the resistant strains) the selection process reaches 935 days.

[0040] On stages E-21 and E-22, 0.5 LD (0.00000030 gr) of bifenthrin are added, with a mortality from 35 to 33% (The same way as in previous stages, E-22 is used to strengthen the growth of resistant strains). On stages E-23 and E-24, thiodicarb is increased to 0.6 LD (0.00000036 gr) with a mortality rate from 30 to 28%. At the end of stage 24 (growth strengthening stage for the resistant strains) the selection process reaches 1050 days.

[0041] On stages E-25 and E-26, 0.7 LD (0.00012 gr) of thiodicarb are added, with a mortality from 25 to 24% (The same way as in previous stages, E-26 is used to strengthen the growth of resistant strains). On stages E-27 and E-28, 0.8 LD (0.00014 gr) of thiodicarb are added, with a mortality rate from 22 to 20%. At the end of stage E-28 (growth strengthening stage for the resistant strains) the selection process reaches 1155 days.

[0042] On stages E-29 and E-30, 0.7 LD (0.00000036 gr) of bifenthrin are added, with a mortality from 20 to 18% (The same way as in previous stages, E-30 is used to strengthen the growth of resistant strains). On stages E-31 and E-32, thiodicarb is increased to 0.8 LD (0.00000042 gr) with a mortality rate from 17 to 15%. At the end of E-32 (growth strengthening stage for the resistant strains) the selection process reaches 1254 days.

[0043] On stages E-33 and E-34, 0.9 LD (0.00016 gr) of thiodicarb are added, with a mortality from 13 to 11% (The same way as in previous stages, E-34 is used to strengthen the growth of resistant strains). On stages E-35 and E-36, 1 LD (0.00018 gr) of thiodicarb are added, with a mortality rate from 10 to 8%. At the end of stage E-36 (growth strengthening stage for the resistant strains) the selection process reaches 1327 days.

[0044] On stages E-37 and E-38, 0.9 LD (0.00000042 gr) of bifenthrin are added, with a mortality from 8 to 6% (The same way as in previous stages, E-38 is used to strengthen the growth of resistant strains). On stages E-39 and E-40, thiodicarb is increased to 1 LD (0.00000048 gr) decreasing mortality to 5%. At the end of stage 40 the selection process reaches 1389 days.
Table No. 2 Comparative Table, Initial and Final Characteristics
SELECTIVE PROCESS CHARACTERISTICS Bacillus megaterium
PARAMETERINITIALFINAL
Ph 7.2 7.2
Humidity 28% 90%
Respiration Aerobic Aerobic
Temperature 52 - 75ºC 25 - 35ºC
Size 1.0 x 3.0 µm 1.0 x 3.0 µm
Resistant to: Organochlorinated, organic phosphorus compounds and mercury 1 LD of thiodicarb and bifenthrin

Bacillus subtillis



[0045] It is a Gram positive bacillus, sporogenous, strictly aerobic, with a thick murein layer, its initial average size is 0.75 µm wide x 0.9 µm long; its growth environmental temperatures go from 15 to 55ºC, B.subtilis' natural habitat is the soil, with great temperature fluctuations. However, the cells from this microorganism are submitted to a phenotypic change when temperature is modified from 30ºC to 45ºC or 80ºC. Its activity is developed within a pH from 4.7 to 5.5, humidity from 70 to 80% and tolerates minimal trace toxic concentrations.

[0046] It is used to produce an antibiotic called bacitracin, acts against Gram negative damaging its cellular membrane and inhibiting the wall formation. Besides the enzyme production such as bacterial amylase, useful in the paper and textile industries, and enzyme used to tan leathers, remove stains and soften meats.

[0047] A microcosmos Model (Kabir and Cols, 1995) was used to isolate these microorganisms. The strains were classified using traditional methods (Numeric taxonomy) and immunochemicals (indirect immunofluorescence-IIF, according to Llewot and Stead, 1991). Pure strains were used from different areas. The growth media used for the B. subtillis is a nutritious media, with 5% of soy-agar maintaining parameters such as pH, minimum oxygen levels, humidity and temperature from its original conditions

Stages performed in the lab for b. subtillis



[0048] Beginning with habitat conditions obtained in the adaptation to organophosphate, organochlorine compounds and mercury from the microorganisms at the E-38 pH: 7.2, Humidity: 36%, thermoresistant and aerobic. These conditions are the starting point to adapt the strain to thiodicarb and bifenthrin.

[0049] To adapt this microorganism, 40 stages were performed, beginning the process with the conventional growth media. During stages E-1 and E-2 (Stage 1 and Stage 2) 0.1 LD (0.0000175 gr) of thiodicarb. After incubation a slow growth is seen for the first 72 hours. Until day 68 of incubation mortality is at 82% and on stage E-2 at 80%. The remaining 20% is maintained for 10 days under daily observation to continue with the microorganism adaptation. Habitat temperature and humidity are maintained. (On stage 2, the same concentration of thiodicarb is used to strengthen the growth of resistant strains). The resistant strains are transferred to solid growth media with 0.2 LD (0.000035 gr) of thiodicarb (E-3 and E-4, again on E-4 the same concentration of thiodicarb is used to strengthen the growth of resistant strains) with a mortality rate from 75 to 72%. At the end of stage E-4, organisms have been selected for 294 days.

[0050] Stages E-5 and E-6 are submitted to the first addition of bifenthrin; 0.1 LD (0.00000006 gr) of bifenthrin are added, with a mortality rate from 72 to 71% (in the same way as the previous case, E-6 is used to strengthen the growth of resistant strains). On stages E-7 and E-8 bifenthrin is increased to 0.2 LD (0.00000012 gr) with a mortality from 70 to 68%. At the end of E-8 (growth strengthening stage for the resistant strains) the adaptation process reaches 570 days.

[0051] On stages E-9 and E-10, 0.3 LD (0.000053 gr) of thiodicarb are added, with a mortality from 66 to 63%. (The same way as in previous stages, E-10 is used to strengthen the growth of resistant strains). On stages E-11 and E-12 0.4 LD (0.000070 gr) of thiodicarb are added, with a mortality rate from 64 to 62%. At the end of stage E-12 (growth strengthening stage for the resistant strains) the selection process reaches 827 days.

[0052] On stages E-13 and E-14, 0.3 LD (0.00000018 gr) of bifenthrin are added, with a mortality of 65% (The same way as in previous stages, E-14 is used to strengthen the growth of resistant strains). On stages E-15 and E-16, thiodicarb is increased to 0.4 LD (0.00000024 gr) with a mortality from 64 to 60%. At the end of stage 16 (growth strengthening stage for the resistant strains) the selection process reaches 1071 days.

[0053] On stages E-17 and E-18, 0.5 LD (0.000087 gr) of thiodicarb are added, with a mortality from 59 to 54% (The same way as in previous stages, E-18 is used to strengthen the growth of resistant strains). On stages E-19 and E-20, 0.6 LD (0.00011 gr) of thiodicarb are added, with a mortality rate from 52 to 50%. At the end of stage E-20 (growth strengthening stage for the resistant strains) the selection process reaches 1283 days.

[0054] On stages E-21 and E-22, 0.5 LD (0.00000030 gr) of bifenthrin are added, with a mortality from 47 to 45% (The same way as in previous stages, E-22 is used to strengthen the growth of resistant strains). On stages E-23 and E-24, thiodicarb is increased to 0.6 LD (0.00000036 gr) with a mortality rate from 40 to 35%. At the end of stage 24 (growth strengthening stage for the resistant strains) the selection process reaches 1458 days.

[0055] On stages E-25 and E-26, 0.7 LD (0.00012 gr) of thiodicarb are added, with a mortality from 35 to 30% (The same way as in previous stages, E-26 is used to strengthen the growth of resistant strains). On stages E-27 and E-28, 0.8 LD (0.00014 gr) of thiodicarb are added, with a mortality rate from 28 to 25%. At the end of stage E-28 (growth strengthening stage for the resistant strains) the selection process reaches 1581 days.

[0056] On stages E-29 and E-30, 0.7 LD (0.00000036 gr) of bifenthrin are added, with a mortality from 25 to 23% (The same way as in previous stages, E-30 is used to strengthen the growth of resistant strains). On stages E-31 and E-32, thiodicarb is increased to 0.8 LD (0.00000042 gr) with a mortality rate from 20 to 18%. At the end of E-32 (growth strengthening stage for the resistant strains) the selection process reaches 1704 days.

[0057] On stages E-33 and E-34, 0.9 LD (0.00016 gr) of thiodicarb are added, with a mortality from 18 to 15% (The same way as in previous stages, E-34 is used to strengthen the growth of resistant strains). On stages E-35 and E-36, 1 LD (0.00018 gr) of thiodicarb are added, with a mortality rate from 12 to 9%. At the end of stage E-36 (growth strengthening stage for the resistant strains) the selection process reaches 1327 days.

[0058] On stages E-37 and E-38, 0.9 LD (0.00000042 gr) of bifenthrin are added, with a mortality from 8 to 6% (The same way as in previous stages, E-38 is used to strengthen the growth of resistant strains). On stages E-39 and E-40, thiodicarb is increased to 1 LD (0.00000048 gr) decreasing mortality to 5%. At the end of stage 40 the selection process reaches 1882 days; 5.6 years of research.
Table No. 3. Comparative Table, Initial and Final Characteristics
DEVELOPMENT CHARACTERISTICS Bacillus Subtillis
PARAMETERINITIALFINAL
pH 4.5 - 5.5 7.2
Humidity 80% 36%
Respiration Aerobic Aerobic
Temperature Thermoresistant Thermoresistant
Resistant to: Organochlorinated, organic phosphorus compounds and mercury 1 LD of thiodicarb and bifenthrin
Size 0.9 x 1.3 µm 0.9 x 1.2 µm
Bio degradation time 120 days 35 days


[0059] At the end of the adaptation process, the microorganism shows normal activity in said media with phosphorus (P2O5) traces in its cellular membrane.

[0060] Also shows that the organic material formed by 80% of carbohydrates stabilizes in an average of 35 days unlike the wild microorganism that takes 120 days.

Bacillus thuringiensis



[0061] It is a non-pathogenic Gram positive bacilli, sporogenous, facultative anaerobic, its initial average size is 1.2 µm wide x 4.8 µm long, its optimal environmental growth temperature varies from 10 to 45ºC, its cellular membrane is completely smooth and its activity is developed within a pH of 6.5 to 7.2 with 60% humidity.

[0062] The B. thuringiensis cells form crystalline appositions visible with an optical microscope. These crystalline appositions are formed by protoxins that contain d-endotoxins, with pesticide activity specific against insects. Some varieties of Bacillus thuringiensis can also produce another toxin, the b-exotoxin. It is a toxin produced during vegetative growth which is a nucleotide byproduct from the adenine that works as an RNA polymerase inhibitor. But the use of this toxin is not allowed in some countries because it is also toxic to mammals.

[0063] The growth media for the B. thuringiensis is a nutritious media, with 5% of soy-agar maintaining parameters such as pH, minimum oxygen levels, humidity and temperature from its original conditions.

Stages performed in the lab for B. thuringiensis



[0064] Beginning with habitat conditions obtained in the adaptation to organophosphate, organochlorine compounds and mercury from the microorganisms at the E-38 pH: 7.2, Humidity: 52%, thermoresistant (74ºC), pH 7.0 and facultative anaerobic. These conditions are the starting point to adapt the strain to thiodicarb and bifenthrin. During all stages a cold light lamp will be used to adapt the microorganism to sunlight.

[0065] To adapt this microorganism, 48 stages were performed, beginning the process with the conventional growth media. During stages E-1 and E-2 (Stage 1 and Stage 2) 0.1 LD (0.0000175 gr) of thiodicarb. After incubation a slow growth is seen for the first 72 hours. Until day 45 of incubation mortality is from 72 to 70% and on stage E-2 at 80%. The remaining 30% is maintained for 10 days under daily observation to continue with the microorganism adaptation. Habitat temperature and humidity are maintained. (On stage 2, the same concentration of thiodicarb is used to strengthen the growth of resistant strains). The resistant strains are transferred to solid growth media with 0.2 LD (0.000035 gr) of thiodicarb (E-3 and E-4, again on E-4 the same concentration of thiodicarb is used to strengthen the growth of resistant strains) with a mortality rate from 70 to 68%. At the end of stage E-4, organisms have been selected for 282 days.

[0066] Stages E-5 and E-6 are submitted to the first addition of bifenthrin; 0.1 LD (0.00000006 gr) of bifenthrin are added, with a mortality rate from 68 to 65% (in the same way as the previous case, E-6 is used to strengthen the growth of resistant strains). On stages E-7 and E-8 bifenthrin is increased to 0.2 LD (0.00000012 gr) with a mortality from 64 to 63%. At the end of E-8 (growth strengthening stage for the resistant strains) the adaptation process reaches 565 days.

[0067] On stages E-9 and E-10, 0.3 LD (0.000053 gr) of thiodicarb are added, with a mortality from 65 to 61%. (The same way as in previous stages, E-10 is used to strengthen the growth of resistant strains). On stages E-11 and E-12 0.4 LD (0.000070 gr) of thiodicarb are added, with a mortality rate from 57 to 55%. At the end of stage E-12 (growth strengthening stage for the resistant strains) the selection process reaches 822 days.

[0068] On stages E-13 and E-14, 0.3 LD (0.00000018 gr) of bifenthrin are added, with a mortality from 57 to 55% (The same way as in previous stages, E-14 is used to strengthen the growth of resistant strains). On stages E-15 and E-16, thiodicarb is increased to 0.4 LD (0.00000024 gr) with a mortality from 52 to 50%. At the end of stage 16 (growth strengthening stage for the resistant strains) the selection process reaches 1064 days.

[0069] On stages E-17 and E-18, 0.5 LD (0.000087 gr) of thiodicarb are added, with a mortality from 52 to 50% (The same way as in previous stages, E-18 is used to strengthen the growth of resistant strains). On stages E-19 and E-20, 0.6 LD (0.00011 gr) of thiodicarb are added, with a mortality rate from 50 to 45%. At the end of stage E-20 (growth strengthening stage for the resistant strains) the selection process reaches 1280 days.

[0070] On stages E-21 and E-22, 0.5 LD (0.00000030 gr) of bifenthrin are added, with a mortality from 50 to 45% (The same way as in previous stages, E-22 is used to strengthen the growth of resistant strains). On stages E-23 and E-24, thiodicarb is increased to 0.6 LD (0.00000036 gr) with a mortality rate from 44 to 42%. At the end of stage 24 (growth strengthening stage for the resistant strains) the selection process reaches 1467 days.

[0071] On stages E-25 and E-26, 0.7 LD (0.00012 gr) of thiodicarb are added, with a mortality from 44 to 40% (The same way as in previous stages, E-26 is used to strengthen the growth of resistant strains). On stages E-27 and E-28, 0.8 LD (0.00014 gr) of thiodicarb are added, with a mortality rate from 36 to 35%. At the end of stage E-28 (growth strengthening stage for the resistant strains) the selection process reaches 1633 days of adaptation.

[0072] On stages E-29 and E-30, 0.7 LD (0.00000036 gr) of bifenthrin are added, with a mortality from 36 to 33% (The same way as in previous stages, E-30 is used to strengthen the growth of resistant strains). On stages E-31 and E-32, thiodicarb is increased to 0.8 LD (0.00000042 gr) with a mortality rate from 30 to 28%. At the end of E-32 (growth strengthening stage for the resistant strains) the selection process reaches 1777 days.

[0073] On stages E-33 and E-34, 0.9 LD (0.00016 gr) of thiodicarb are added, with a mortality of 30% (The same way as in previous stages, E-34 is used to strengthen the growth of resistant strains). On stages E-35 and E-36, 1 LD (0.00018 gr) of thiodicarb are added, with a mortality rate of 25%. At the end of stage E-36 (growth strengthening stage for the resistant strains) the selection process reaches 1909 days of adaptation.

[0074] On stages E-37 and E-38, 0.9 LD (0.00000042 gr) of bifenthrin are added, with a mortality from 30 to 28% (The same way as in previous stages, E-38 is used to strengthen the growth of resistant strains). On stages E-39 and E-40, thiodicarb is increased to 1 LD (0.00000048 gr) decreasing mortality from 18 to 17%. At the end of stage 40 the selection process reaches 2015 days.

[0075] On stages E-41 and E-42, 1.1 LD (0.00020 gr) of thiodicarb are added, with a mortality from 18 to 17% (The same way as in previous stages, E-42 is used to strengthen the growth of resistant strains). On stages E-43 and E-44, 1.2 LD (0.00022 gr) of thiodicarb are added, with a mortality rate on both stages of 14%. At the end of stage E-44 (growth strengthening stage for the resistant strains) the selection process reaches 2016 days of adaptation.

[0076] On stages E-45 and E-46, 1.1 LD (0.00000054 gr) of bifenthrin are added, with a mortality from 15 to 12% (The same way as in previous stages, E-46 is used to strengthen the growth of resistant strains). On stages E-47 and E-48, thiodicarb is increased to 1.2 LD (0.00000060 gr) decreasing mortality from 10 to 7%. At the end of stage 48 (growth strengthening stage for the resistant strains) the selection process reaches 2182 days of adaptation.
Table No. 4. Comparative Table, Initial and Final Characteristics
DEVELOPMENT CHARACTERISTICS B. Thuringiensis
PARAMETERINITIALFINAL
pH 4.5 - 5.5 7.2
Humidity 10% 100%
Respiration Anaerobic Aerobic
Temperature 30-37 ºC 25 ºC
Resistant to: Organochlorinated, organophosphorus compounds and mercury 1.2 LD of thiodicarb and bifenthrin
Size 1.0 x 3.0 µm 1.0 x 3.0 µm
Biodegradation time 120 days 35 Days

Clostridium pasteuranium



[0077] It is a Gram positive bacillus, anaerobic and heterotrophic, its natural habitat is soil, it has a phosphorylation and electron transportation mechanism, for which the ATP is obtained through phosphorylation at a soil level.

[0078] It is optimal development is within a temperature range of 20 to 30ºC. Mobile, moves using a flagellum, it is chemoorganotrophic. This bacillus is facultative anaerobic.

Stages performed in the lab using clostridium pasteuranium



[0079] Beginning with habitat conditions obtained for the adaptation to organophosphorus, organochlorinated compounds and mercury for the microorganism in the E-33 pH: 7.2, Humidity: 40%, thermoresistant. These conditions are the starting point to adapt the strain to the new pollutants: thiodicarb and bifenthrin.

[0080] To adapt this microorganism, 40 stages were performed starting the processes with the conventional growth media. During stages E-1 and E-2 (Stage 1 and Stage 2) 0.1 LD (0.0000175 gr) of thiodicarb are added to the growth media. After incubation a slow growth is recorded for 72 hours. Until incubation day 72 with a mortality of 87% for E-1 that decreases to 84% for E-2. The remaining 16% is under daily observation for 10 days to continue with the adaptation of this microorganism. Habitat humidity and temperature are maintained. (On stage 2, the same concentration of thiodicarb is used to strengthen the growth of resistant strains). The resistant strains are transferred to solid growth media with 0.2 LD (0.000035 gr) of thiodicarb (E-3 and E-4, again on E-4 the same concentration of thiodicarb is used to strengthen the growth of resistant strains) with a mortality between 66 to 62%. At the end of stage E-4, organisms have been selected for 281 days.

[0081] Stages E-5 and E-6 are submitted to the first addition of bifenthrin; 0.1 LD (0.00000006 gr) of bifenthrin are added, with a mortality from 81 to 77% (in the same way as the previous case, E-6 is used to strengthen the growth of resistant strains). On stages E-7 and E-8 bifenthrin is increased to 0.2 LD (0.00000012 gr) with a mortality from 68 to 74 to 70%. At the end of E-8 (growth strengthening stage for the resistant strains) the adaptation process reaches 552 days.

[0082] On stages E-9 and E-10, 0.3 LD (0.000053 gr) of thiodicarb are added, with a mortality from 70 to 69%. (The same way as in previous stages, E-10 is used to strengthen the growth of resistant strains). On stages E-11 and E-12 0.4 LD (0.000070 gr) of thiodicarb are added, with a mortality rate from 60 to 67%. At the end of stage E-12 (growth strengthening stage for the resistant strains) the selection process reaches 803 days.

[0083] On stages E-13 and E-14, 0.3 LD (0.00000018 gr) of bifenthrin are added, with a mortality from 66 to 63% (The same way as in previous stages, E-14 is used to strengthen the growth of resistant strains). On stages E-15 and E-16, thiodicarb is increased to 0.4 LD (0.00000024 gr) with a mortality from 60 to 58%. At the end of stage 16 (growth strengthening stage for the resistant strains) the selection process reaches 1027 days.

[0084] On stages E-17 and E-18, 0.5 LD (0.000087 gr) of thiodicarb are added, with a mortality from 58 to 55% (The same way as in previous stages, E-18 is used to strengthen the growth of resistant strains). On stages E-19 and E-20, 0.6 LD (0.00011 gr) of thiodicarb are added, with a mortality rate from 55 to 51%. At the end of stage E-20 (growth strengthening stage for the resistant strains) the selection process reaches 1121 days.

[0085] On stages E-21 and E-22, 0.5 LD (0.00000030 gr) of bifenthrin are added, with a mortality from 48 to 46% (The same way as in previous stages, E-22 is used to strengthen the growth of resistant strains). On stages E-23 and E-24, thiodicarb is increased to 0.6 LD (0.00000036 gr) with a mortality rate from 40 to 38%. At the end of stage 24 (growth strengthening stage for the resistant strains) the process reaches 1362 days of work.

[0086] On stages E-25 and E-26, 0.7 LD (0.00012 gr) of thiodicarb are added, with a mortality from 37 to 35% (The same way as in previous stages, E-26 is used to strengthen the growth of resistant strains). On stages E-27 and E-28, 0.8 LD (0.00014 gr) of thiodicarb are added, with a mortality rate from 33 to 30%. At the end of stage E-28 (growth strengthening stage for the resistant strains) the adaptation process reaches 1506 days.

[0087] On stages E-29 and E-30, 0.7 LD (0.00000036 gr) of bifenthrin are added, with a mortality from 29 to 25% (The same way as in previous stages, E-30 is used to strengthen the growth of resistant strains). On stages E-31 and E-32, thiodicarb is increased to 0.8 LD (0.00000042 gr) with a mortality rate from 22 to 18%. At the end of stage 32 (growth strengthening stage for the resistant strains) the process reaches 1631 days of work.

[0088] On stages E-33 and E-34, 0.9 LD (0.00016 gr) of thiodicarb are added, with a mortality from 18 to 16% (The same way as in previous stages, E-34 is used to strengthen the growth of resistant strains). On stages E-35 and E-36, 1 LD (0.00018 gr) of thiodicarb are added, with a mortality rate from 13 to 12%. At the end of stage E-36 (growth strengthening stage for the resistant strains) the adaptation process reaches 1701 days.

[0089] On stages E-37 and E-38, 0.9 LD (0.00000042 gr) of bifenthrin are added, with a mortality of 10% (The same way as in previous stages, E-38 is used to strengthen the growth of resistant strains). On stages E-39 and E-40, thiodicarb is increased to 1 LD (0.00000048 gr) with a mortality rate from 8 to 5%. At the end of stage 40 (growth strengthening stage for the resistant strains) the selection process reaches 1777 days of work.
Table No. 5. Comparative Table, Initial and Final Characteristics
SELECTIVE PROCESS CHARACTERISTICS C. Pasteuranium
PARAMETERINITIALFINAL
pH 6.5 - 7.2 7.5
Humidity 10% 100%
Respiration Aerobic Aerobic
Temperature 25 - 40°C 25ºC
Size 1.0 x 3.0 µm 1.3 x 3.5 µm
Resistant to: Organochlorinated, organic phosphorus compounds and mercury 1 DL of thiodicarb and bifenthrin

Rhizobium so



[0090] It is a facultative anaerobic bacillus, grows in low oxygen concentrations, it's natural habitat is soil, swamp soil and water; it is a chemoorganotrophic bacteria. They have a complex cellular wall formed by an external membrane and a peptidoglycan internal layer that contains muramic acid and murein, which makes it a N2 fixing bacteria that absorbs it from the atmosphere and stores it in its murein layer. Its optimal development is within a temperature from 20 to 30ºC, humidity from 25 to 40% and an acid pH. Mobile.

Stages performed in the lab using con Rhizobium sp



[0091] Beginning with habitat conditions: pH: 2.0-4.5, Humidity: 25- 40%, temperature 20-30 ºC. Facultative anaerobic. These conditions are the starting point to adapt the strain to thiodicarb and bifenthrin. During all stages, we will be using a cold light lamp to adapt the microorganism to sunlight.

[0092] To adapt this microorganism, 40 stages were performed, beginning the process with the conventional growth media. During stages E-1 and E-2 (Stage 1 and Stage 2) 0.1 LD (0.0000175 gr) of thiodicarb are added. After incubation a slow growth is seen for the first 72 hours. Until day 68 of the incubation with mortality from 63 to 62%. The remaining 38% is kept under daily surveillance for 10 days to continue with the adaptation of the microorganism. Temperature and humidity of its habitat are maintained.(On stage 2, the same concentration of thiodicarb is used to strengthen the growth of resistant strains). The resistant strains are transferred to solid growth media with 0.2 LD (0.000035 gr) of thiodicarb (E-3 and E-4, again on E-4 the same concentration of tiodicarb is used to strengthen the growth of resistant strains) with a 60% mortality rate. At the end of stage E-4, organisms have been selected for 253 days.

[0093] Stages E-5 and E-6 are submitted to the first addition of bifenthrin; 0.1 LD (0.00000006 gr) of bifenthrin are added, with a mortality from 62 to 60% (in the same way as the previous case, E-6 is used to strengthen the growth of resistant strains). On stages E-7 and E-8 bifenthrin is increased to 0.2 LD (0.00000012 gr) with a mortality from 58 to 55%. At the end of E-8 (growth strengthening stage for the resistant strains) the adaptation process reaches 501 days.

[0094] On stages E-9 and E-10, 0.3 LD (0.000053 gr) of thiodicarb are added, with a mortality from 55 to 53%. (The same way as in previous stages, E-10 is used to strengthen the growth of resistant strains). On stages E-11 and E-12 0.4 LD (0.000070 gr) of thiodicarb are added, with a mortality rate from 51 to 49%. At the end of stage E-12 (growth strengthening stage for the resistant strains) the selection process reaches 727 days.

[0095] On stages E-13 and E-14, 0.3 LD (0.00000018 gr) of bifenthrin are added, with a mortality from 49 to 47% (The same way as in previous stages, E-14 is used to strengthen the growth of resistant strains). On stages E-15 and E-16, thiodicarb is increased to 0.4 LD (0.00000024 gr) with a mortality from 44 to 42%. At the end of stage 16 (growth strengthening stage for the resistant strains) the selection process reaches 936 days.

[0096] On stages E-17 and E-18, 0.5 LD (0.000087 gr) of thiodicarb are added, with a mortality from 41 to 40% (The same way as in previous stages, E-18 is used to strengthen the growth of resistant strains). On stages E-19 and E-20, 0.6 LD (0.00011 gr) of thiodicarb are added, with a mortality rate from 38 to 37%. At the end of stage E-20 (growth strengthening stage for the resistant strains) the selection process reaches 1122 days.

[0097] On stages E-21 and E-22, 0.5 LD (0.00000030 gr) of bifenthrin are added, with a mortality from 36 to 35% (The same way as in previous stages, E-22 is used to strengthen the growth of resistant strains). On stages E-23 and E-24, thiodicarb is increased to 0.6 LD (0.00000036 gr) with a mortality rate from 33 to 31%. At the end of stage 24 (growth strengthening stage for the resistant strains) the selection process reaches 1268 days.

[0098] On stages E-25 and E-26, 0.7 LD (0.00012 gr) of thiodicarb are added, with a mortality from 30 to 28% (The same way as in previous stages, E-26 is used to strengthen the growth of resistant strains). On stages E-27 and E-28, 0.8 LD (0.00014 gr) of thiodicarb are added, with a mortality rate from 25 to 23%. At the end of stage E-28 (growth strengthening stage for the resistant strains) the selection process reaches 1393 days.

[0099] On stages E-29 and E-30, 0.7 LD (0.00000036 gr) of bifenthrin are added, with a mortality from 21 to 18% (The same way as in previous stages, E-30 is used to strengthen the growth of resistant strains). On stages E-31 and E-32, thiodicarb is increased to 0.8 LD (0.00000042 gr) with a mortality rate from 15 to 14%. At the end of E-32 (growth strengthening stage for the resistant strains) the selection process reaches 1505 days.

[0100] On stages E-33 and E-34, 0.9 LD (0.00016 gr) of thiodicarb are added, with a mortality from 25 to 22% (The same way as in previous stages, E-34 is used to strengthen the growth of resistant strains). On stages E-35 and E-36, 1 LD (0.00018 gr) of thiodicarb are added, with a mortality rate from 20 to 18%. At the end of stage E-36 (growth strengthening stage for the resistant strains) the adaptation process reaches 1601 days.

[0101] On stages E-37 and E-38, 0.9 LD (0.00000042 gr) of bifenthrin are added, with a mortality from 16 to 14% (The same way as in previous stages, E-38 is used to strengthen the growth of resistant strains). On stages E-39 and E-40, thiodicarb is increased to 1 LD (0.00000048 gr) maintaining the mortality from 10 to 8%. At the end of stage 40 the selection process reaches 1677 days of work.
Table No. 6. Comparative Table, Initial and Final Characteristics
DEVELOPMENT CHARACTERISTICS Rhizobium sp
PARAMETERINITIALFINAL
pH 4.5 - 7.2 7.2
Humidity 10% 100%
Respiration Anaerobic Aerobic
Temperature 30 - 37 ºC 25ºC
Size 1.0 x 3.0 µm 1.0 x 3.0 µm
Resistant to: Organochlorinated, organic phosphorus and mercury compounds 1 DL of thiodicarb and bifenthrin


[0102] The microorganism subjected to poisoning with thiodicarb and bifenthrin traces shows normal activity in said media, finding traces of nitrogen in its cellular membrane.

Maintenance of the strains resistant to carbamate traces contained in thiodicarb and pyrethroid traces contained in bifenthrin



[0103] During the development of this research, performed in a term over 5 years long, the strains were submitted to a physical media change, adapting them to withstand increasing concentrations of thiodicarb and bifenthrin.

[0104] Once the microorganisms adapted to the media were obtained, the strain purity is verified taking a 0.1 ml aliquot from the microorganism culture suspension and seed a tryptic soy agar dish (TSA) incubated at 52ºC +/- 2ºC, verifying its mobility with malachite green Gram.

[0105] After determining the strain purity, a suspension of this microorganism is made in a TSA dish incubated from 48 to 72 hours at 52ºC +/- 2ºC, after that, a sample is taken using a sterile swab and 100 ml of liquid media containing 0.01 LD of bifenthrin and/or 0.01 LD of thiodicarb are seeded, it is stirred for 4 hours at 37ºC and 170 rpm; after that, the biomass is diluted in 50 ml of the media contained in different lyoprotectant solutions (used to prepare microorganisms for lyophilization), the media is homogenized and the CFU/ml is determined. This inoculum is taken to the incubator and is periodically seeded in the same culture media. This procedure is performed in intervals from 72 hours to 120 hours; all these procedures are performed periodically up to the date. The maintenance time in the same conditions is used to perform general readings.

[0106] These strains are kept in the active culture media adding a previously sterilized mineral oil, this way the required humidity is guaranteed.

References



[0107] 

Castro, L. 1997. Mixture for the biodegradation of organic matter consisting of a specific growth media and microorganisms adapted to air and the seeding process. Patent No. 26662 issued by the Economic development Ministry of Colombia.

Castro, L. 2006. Composite of microorganisms adapted to biodegrade the Organic portion contained in solid residues and the preparation process. Patent No. PA/2006/003777, issued by the IMPI. Mexico. (Issued April 27, 2017)

Orozco J. C, Martinez P. Test of the seeding of nitrogen-fixing non-symbiotic microorganisms isolated from the rhizosphere of Pinus patula in Colombia. Alexander von Humboldt Biological Research Institute, La Florida Forest Station, Cota. BOSQUE 30(2): 70-77, Cundinamarca-Colombia. 2009.

Cuervo J. Isolation and Characterization of the Bacillus spp as biological nitrogen fixers and phosphate solubilizers in two samples of commercial biofertilizers. Degree work. Agricultural and livestock microbiology. Bogotá-Colombia, 2010.

Kabir, M. and D. Faure. 1995. Identification of Azospirillum by oligonucleotide probes after isolation from soil and Sourghum rizoplan contaminated or not by the parasitic plant Siriga. Advances in Applied Microbiology. 35. 195-253.

Kabir, M.; Faure, D.; Heulin, T.; Achoawk, W.; and R. Bally. 1995. Oligonucleotide probes based on 165 Rrna sequences for the identification of four Azospirillum species. Can. J. Microbiol. 41: 1081-1087.

Validation of the Publication of New Names and New Combinations. Previously Effectively Published Outside the IJSB. List no. 51. International Journal of Systematic Bacteriology, 1994.




Claims

1. A consortium of microorganisms resistant to 1 to 1.3 (LD) of the lethal dose of thiodicarb and bifenthrin. This consortium is able to be seeded in liquid, foliar, edaphic and/or irrigation systems production, characterized because it is formed of a mixture of the following microorganisms: Clostridium pasteuranium, Bacillus thuringiensis, Bacillus megaterium, Bacillus subtillis, Azotobacter vinelandil and Rhizobium sp
 
2. The consortium complies with claim 1 resistant to 1 to 1.3 lethal dose (LD) of thiodicarb and bifenthrin, characterized because such organisms are found on the following percentages: Clostridium pasteuranium 15%, Bacillus thuringiensis 20%, Bacillus megaterium 20%, Bacillus subtillis 15%, Azotobacter vinelandii 15% and Rhizobium sp 15%.
 
3. The consortium complies with claims 1 and 2 resistant to concentrations of 1 to 1.3 of the lethal dose (LD) of thiodicarb and bifenthrin, characterized because the ingredients of the growth media for the compound and the conditions are: 40% conventional growth media, 10% of soy agar, 10% of malt agar and 40% of sterilized water. The conditions are: pH between 7.0 and 7.2, humidity 100% and a temperature between 22 and 28°C.
 
4. The consortium complies with claim 1 resistant to concentrations of 1 to 1.3 of the lethal dose (LD) of thiodicarb and bifenthrin, characterized by its use in the production of liquid fertilizers or directly to the crop irrigation system.
 
5. The consortium complies with claims 1, 2, 3 and 4 resistant to concentrations of 1 to 1.3 of the lethal dose (LD) of thiodicarb and bifenthrin, characterized because when it is applied to liquid fertilizers containing them, they are resistant and withstand sunlight guaranteeing soil repopulation.
 
6. The consortium complies with claims 1 resistant to concentrations of 1 to 1.3 of the lethal dose (LD) of thiodicarb and bifenthrin, comprised of bacteria. These microorganisms are not necessarily together, sometimes there are greater concentrations of some, according to the agricultural requirements of the crop.
 
7. The consortium complies with claims 1, 2, 3 and 5 resistant to concentrations of 1 to 1.3 of the lethal dose (LD) of thiodicarb and bifenthrin acting as biofertilizer, biopesticide and bio-fungicide on crops either edaphic and foliar.
 





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