Method for making resistant to thiodicarb (carbamate) and bifenthrin (pyrethroid) a consortium of fungi that solubilise phosphorous and antagonise certain pathogens, for use in liquid biofertilisers for foliar and/or soil application

Abstract

The present invention provides a method for making a consortium of fungi resistant to traces of carbamates contained in thiodicarb and to traces of synthetic pyrethroids contained in bifenthrin. During the course of this research, carried out over more than five years, the strains were subjected to a change in physical medium, adapting same to tolerate increasing concentrations of thiodicarb and bifenthrin. This compound enriches and increases the concentration of beneficial microorganisms, generating high-quality liquid biological fertilisers suitable for use in agricultural production, in land recovery and conservation, under the parameters established in sustainable organic farming, which seeks to conserve, recover and use nature or the environment without generating the least negative impact.

Claims

1. A consortium of microorganisms including fungi and bacteria, wherein the microorganisms of the consortium are initially exposed to a series of increasing fractions of a lethal dose of thiodicarb, subsequently exposed to a series of increasing fractions of a lethal dose of bifenthrin, wherein subsequent to the exposures of thiodicarb and bifenthrin, the microorganisms of the consortium are resistant to 1 to 1.3 times the lethal dose of thiodicarb and bifenthrin, wherein the consortium is able to be seeded in liquid, foliar, edaphic or irrigation systems, wherein the consortium was deposited with the National Center of Genetic Resources (CM-CNRG) under accession number CM-CNRG TB46.

2. The consortium according to claim 1, wherein the consortium is operable to act as any of a biofertilizer, biopesticide and biofungicide on crops, either under edaphic or foliar conditions.

3. The consortium according to claim 1, wherein the consortium is operable to grow and conduct metabolic processes in a presence of sunlight, either under edaphic or foliar conditions.

4. A consortium of microorganisms including fungi and bacteria, wherein the microorganisms of the consortium are exposed to increasing first fractions of a lethal dose of thiodicarb, subsequently exposed to increasing first fractions of a lethal dose of bifenthrin, subsequently exposed to increasing second fractions of a lethal dose of thiodicarb, wherein the second fractions of a lethal dose of thiodicarb are greater than the first fractions of a lethal dose of thiodicarb, subsequently exposed to increasing second fractions of a lethal dose of bifenthrin, wherein the second fractions of a lethal dose of bifenthrin are greater than the first fractions of a lethal dose of bifenthrin, subsequently exposed to increasing third fractions of a lethal dose of thiodicarb, wherein the third fractions of a lethal dose of thiodicarb are greater than the second fractions of a lethal dose of thiodicarb, subsequently exposed to increasing third fractions of a lethal dose of bifenthrin, wherein the third fractions of a lethal dose of bifenthrin are greater than the second fractions of a lethal dose of bifenthrin, subsequently exposed to increasing fourth fractions of a lethal dose of thiodicarb, wherein the fourth fractions of a lethal dose of thiodicarb are greater than the third fractions of a lethal dose of thiodicarb, subsequently exposed to increasing fourth fractions of a lethal dose of bifenthrin, wherein the fourth fractions of a lethal dose of bifenthrin are greater than the third fractions of a lethal dose of bifenthrin, subsequently exposed to increasing fifth fractions of a lethal dose of thiodicarb, wherein the fifth fractions of a lethal dose of thiodicarb are greater than the fourth fractions of a lethal dose of thiodicarb, and subsequently exposed to increasing fifth fractions of a lethal dose of bifenthrin, wherein the fifth fractions of a lethal dose of bifenthrin are greater than the fourth fractions of a lethal dose of bifenthrin, wherein subsequent to the first through fifth exposures of thiodicarb and the first through fifth exposures of bifenthrin, the microorganisms of the consortium are resistant to a lethal dose of thiodicarb and bifenthrin, wherein the consortium is operable to be seeded in liquid, foliar, edaphic or irrigation systems, wherein the consortium was deposited with the National Center of Genetic Resources (CM-CNRG) under accession number CM-CNRG TB46.

Description

PROCEDURE DESCRIPTION

(1) During the entire research, for each of the fungi strains and on each of the stages, the following were performed:

(2) a. Modifications to the growth media for each microorganism. Coloring to determine its size, form, cellular morphology, Gram staining and methyl violet to differentiate and view the hyphae or propagulus, mycelium, internal wall and cellular wall.

(3) b. Growth charts and colony forming unit CFU/ml count per milliliter for the scheduled and defined times for this research.

(4) c. The mycological strains were subject to direct lighting on each of the stages; starting with a soft light and increasing the intensity gradually through the stages; obtaining the desired adaptation.

(5) d. Adaptation based in the humidity increase and temperature decrease.

Materials and Methods

(6) Growth media: Liquids: Nutritious media. Solids: Agar, gel or silica gel, Nutrient agar, meat extract, yeast extract, sabouraud broth, malt extract, rice substrate, corn flour and some minerals.

(7) Carbon sources: Organic: Peptone and sucrose (4-5%).

(8) Nitrogen source: NH4 and nitrites.

(9) Light source: Artificial light (low and medium intensity lamp).

(10) Other growth elements: Water, hydrogen, oxygen, nitrogen, sulphur, phosphorous, potassium, magnesium, calcium, iron and sodium.

(11) Trace elements: Cobalt, zinc, potassium, magnesium, molybdenum, sulphur, copper, manganese, carbamate (thiodicarb) and pyrethroid (bifenthrin).

(12) Strains of sulphur sulubilizer fungi, antagonist to some pathogens: a) Paecilomyces sp. b) Beauveria bassiana c) Trichoderma harzianum d) Metarhizium anisopliae
Preparation for Stage Development
1. Glass material is sterilized in an autoclave.
2. The required amount of growth media is prepared with 3% rice broth (P/V), and 9% of a solution of molasses, sterilized at 121° C. for 15 minutes at a pressure of 15 lb.
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 25° C. for 72 hours with cold light.
4. From the previous suspension with a known concentration, (107) the growth media is seeded with 1 ml, the pH adjustment is performed at 5.5 with a sterile 10 citric acid solution.
5. Stir at 110 rpm and 27 OC for 7 days.
6. The process is monitored controlling the variables, mainly the pH and biomass concentration, adjusting the pH to 5.5 during the entire process.
7. On the fourth day of the process, the foam formation is controlled with 1% soy oil and the stirring speed.
Methodology
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.sup.−1 and 10.sup.−2 solutions are performed, and each solution is homogenized.
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.
c. Dishes with thiodicarb and Bifenthrin are placed inside the incubator at 25° C. for 72 hours with a cold light lamp.
d. Inoculated dishes are incubated for 15 days, the product concentration impact is determined on mycelial growth, sporulation and germination (12 hours of incubation).
e. The residual impact of the product is reviewed after 7 days in the malt agar, dextrose and PDA agar media, enriched with minerals.
f. If there is no growth over 10%, the process is repeated until a mortality under 90% is obtained.
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 %).
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, later, a suspension of spores is prepared using the colony sweep method, the obtained suspension is placed in 160×20 mm tubes and 30 ml of sterile distilled water is added. After that, the suspension is stirred for 30 seconds in a Vortex tube mixer. The spore concentration is measured in a Newbawer Chamber and the results are reported in conidia per square millimeter.
i. From the 10 Petri dishes for each microorganism, the one with the lowest mortality rate is the one that will continue with the following study.
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.
k. With the data obtained from the mycelial growth and sporulation, the percentage of inhibition of gradual growth is obtained, the data is transformed for its analysis with the expression 2 arc sin, and the sporulation through the equation Log 10(x+10). A two-way classification analysis of variance is applied and later the Turkey test is performed using a 5% probability.

Records

(13) Characteristics for this procedure were recorded during all stages through the following indicators:

(14) a. Growth speed

(15) b. Percentage of inhibition measured in Number of CFU/mm

(16) c. Percentage of poison resistance

Stages Performed on Each Microorganism

(17) 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:

(18) Paecilomyces sp.

(19) It is an entomopathogenic fungi, it can survive on organic matter in the soil and it is always present in the field, mainly in humid areas, it has a burgundy color. The conidiophores are erected, reaching from 400 to 600 μm growing individually (rarely in groups or synnemata) from the horizontal mycelium. Conidias are oval to fusiform from 2.5 to 3 μm long and from 2.0 to 2.2 μm wide.

(20) Reproduction in the Laboratory

(21) It is grown mainly in rice substrate with 47% humidity for 7 days at 28° C.

(22) The growth media is prepared in an Erlenmeyer flask with PDA (Potato dextrose agar). A spore sample is taken from the fungi culture collection from the lab.

(23) It is seeded in sterile Petri dishes, in potato dextrose agar and malt extract to maintain the colony pure. It is allowed to solidify for 2 to 3 minutes. After that, incubated at 24° C.+/−1 for 12-hour periods, alternating light/darkness for 15, 30 to 45 day periods.

(24) Stages Performed in the Lab Using Paecilomyces sp.

(25) During stages E-1 and E-2 (Stage 1 and Stage 2) 0.1 LD (0.0000175 gr) with a mortality rate from 60 to 62%. (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 58 to 60%. At the end of stage E-4, organisms have been selected for 206 days.

(26) 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 55 to 58% (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 51 to 53% mortality rate. At the end of E-8 (growth strengthening stage for the resistant strains) the adaptation process reaches 384 days.

(27) On stages E-9 y E-10, 0.3 LD (0.000053 gr) of thiodicarb are added, with a mortality rate from 50 to 48%. (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 47 to 48%. At the end of stage E-12 (growth strengthening stage for the resistant strains) the selection process reaches 540 days.

(28) On stages E-13 and E-14, 0.3 LD (0.00000018 gr) of bifenthrin are added, with a mortality rate from 45 to 44%. On stages E-15 and E-16, thiodicarb is increased to 0.4 LD (0.00000024 gr) with a 42 to 44% mortality rate. At the end of stage 16 (growth strengthening stage for the resistant strains) the selection process reaches 687 days.

(29) On stages E-17 and E-18, 0.5 LD (0.000087 gr) of thiodicarb are added, with a mortality rate from 40 to 42% (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 42%. At the end of stage E-20 (growth strengthening stage for the resistant strains) the selection process reaches 823 days.

(30) On stages E-21 and E-22, 0.5 LD (0.00000030 gr) of bifenthrin are added, with a mortality rate from 30 to 36% (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 27 to 31%. At the end of stage 24 (growth strengthening stage for the resistant strains) the selection process reaches 935 days.

(31) On stages E-25 and E-26, 0.7 LD (0.00012 gr) of thiodicarb are added, with a mortality rate from 25 to 23% (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 18 to 21%. At the end of stage E-28 (growth strengthening stage for the resistant strains) the selection process reaches 1032 days.

(32) On stages E-29 and E-30, 0.7 LD (0.00000042 gr) of bifenthrin are added, with a mortality rate from 12 to 15% (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 (0.00000048 gr) with a mortality rate from 8 to 10%. At the end of stage 232 (growth strengthening stage for the resistant strains) the selection process reaches 1112 days.

(33) On stages E-33 and E-34, 0.9 LD (0.00016 gr) of thiodicarb are added, with a mortality rate of 8% (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 15%. At the end of stage E-36 (growth strengthening stage for the resistant strains) the selection process reaches 1177 days.

(34) On stages E-37 and E-38, 0.9 LD (0.00016 gr) of bifenthrin are added, with a mortality rate 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 (0.00018 gr) with a mortality rate of 5%. At the end of stage 40 (growth strengthening stage for the resistant strains) the selection process reaches 1223 days.

(35) TABLE-US-00001 TABLE NO. 1 Comparative Table, Initial and Final Characteristics SELECTIVE PROCESS CHARACTERISTICS Paecilomyces sp. PARAMETER INITIAL FINAL Ph 7.0 7.0 Humidity 35% 70% Respiration Aerobic Aerobic Temperature 24 a 28° C. 24 a 28° C. Size 80 to 250 μm in diameter 80 to 250 μm in diameter Resistant to: Undetectable Resistant to 1 LD of thiodicarb and bifenthrin
Beauveria bassiana

(36) It is an ascomycete mytosporic fungi that grows naturally in soils, with heterotrophic eukaryotic entomopathogen mycelium with chitinous cells. Its entomopathogenic ability allows insect parasitation. Its structure is formed by septated hyphae. The oval to fusiform conidia, from 2.5 to 3 μm long and from 2.0 to 2.5nμm wide. Spores are spherical and slightly oval with a whitish color. It grows on PDA in the lab in approximately 7 days at 8° C.

(37) Stages Performed in the Lab Using Beauveria bassiana

(38) Stages begin with the conditions in its natural habitat: pH—7.0 a 7.5, Humidity: 30 to 40% and humidity is increased up to 70% and a Temperature from 28 to 30° C., maintained during all the research term. These conditions are the starting point to adapt the strain to thiodicarb (carbamate) and bifenthrin (pyrethroid). During all stages, we will be using a cold light lamp to adapt the microorganism to sunlight.

(39) To adapt this microorganism, 56 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 rate of 73% (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 73-79% mortality rate. At the end of stage E-4, organisms have been selected for 281 days.

(40) 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 71 to 70% 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 rate from 68 to 65%. At the end of E-8 (growth strengthening stage for the resistant strains) the adaptation process reaches 547 days.

(41) On stages E-9 and E-10, 0.3 LD (0.000053 gr) of thiodicarb are added, with a mortality rate from 66 to 65%. (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 62 to 59%. At the end of stage E-12 (growth strengthening stage for the resistant strains) the selection process reaches 796 days.

(42) On stages E-13 and E-14, 0.3 LD (0.00000018 gr) of bifenthrin are added, with a mortality rate 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 rate from 53 to 50%. At the end of stage 16 (growth strengthening stage for the resistant strains) the selection process reaches 1036 days.

(43) On stages E-17 and E-18, 0.5 LD (0.000087 gr) of thiodicarb are added, with a mortality rate from 51 to 49% (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 47 to 44%. At the end of stage E-20 (growth strengthening stage for the resistant strains) the selection process reaches 1245 days.

(44) On stages E-21 and E-22, 0.5 LD (0.00000030 gr) of bifenthrin are added, with a mortality rate from 43 to 40% (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 selection process reaches 1430 days.

(45) On stages E-25 and E-26, 0.7 LD (0.00012 gr) of thiodicarb are added, with a mortality rate of 23% (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 34 to 33%. At the end of stage E-28 (growth strengthening stage for the resistant strains) the selection process reaches 1595 days.

(46) On stages E-29 and E-30, 0.7 LD (0.00000036 gr) of bifenthrin are added, with a mortality rate from 32 to 30% (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 1729 days.

(47) On stages E-33 and E-34, 0.9 LD (0.00016 gr) of thiodicarb are added, with a mortality rate from 28 to 27% (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 26%. At the end of stage E-36 (growth strengthening stage for the resistant strains) the selection process reaches 1845 days.

(48) On stages E-37 and E-38, 0.9 LD (0.00000042 gr) of bifenthrin are added, with a mortality rate from 27 to 26% (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 rate from 25 to 24%. At the end of stage 40 the selection process reaches 1952 days.

(49) On stages E-41 and E-42, 1.1 LD (0.0002 gr) of thiodicarb are added, with a mortality rate from 23 to 22% (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 of 20%. At the end of stage E-28 (growth strengthening stage for the resistant strains) the selection process reaches 2040 days.

(50) On stages E-45 and E-46, 1.1 LD (0.00000054 gr) of bifenthrin are added, with a mortality rate from 20 to 28% (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 0.8 LD (0.00000060 gr) with a mortality rate from 16 to 14%. At the end of E-48 the selection process reaches 2110 days.

(51) On stages E-49 and E-50, 1.1 LD (0.00024 gr) of thiodicarb are added, with a mortality rate of 14% (The same way as in previous stages, E-50 is used to strengthen the growth of resistant strains). On stages E-51 and E-52, 1.2 LD (0.00026 gr) of thiodicarb are added, with a mortality rate of 11%. At the end of stage E-52 (growth strengthening stage for the resistant strains) the selection process reaches 2198 days.

(52) On stages E-53 and E-54, 1.2 LD (0.00000066 gr) of bifenthrin are added, with a mortality rate of 8% (The same way as in previous stages, E-54 is used to strengthen the growth of resistant strains). On stages E-55 and E-56, thiodicarb is increased to 1.3 LD (0.00000072 gr) with a mortality rate of 8%. At the end of E-56 (growth strengthening stage for the resistant strains) the selection process reaches 2268 days.

(53) TABLE-US-00002 TABLE NO. 2 Comparative Table, Initial and Final Characteristics SELECTIVE PROCESS CHARACTERISTICS Bacillus megaterium PARAMETER INITIAL FINAL Ph 7.0 7.0 Humidity 35% 70% Respiration Aerobic Aerobic Temperature 24-28° C. 24-28° C. Size 1.0 × 3.0 μm 1.0 × 3.0 μm Measurements 80 to 250 μm in diameter 80 to 250 μm in diameter Resistant to: Organochlorinated, organic 1 LD of thiodicarb phosphorus compounds and and bifenthrin mercury
Trichoderma harzianum

(54) It is a fungus that can be found in different organic matter, soils and in different environmental conditions, for which it is easy to adapt and propagate; prefer an acid pH from 4 to 4.5 and high humidity. This fungus generates toxins and antibiotics. Its mycelium has a whitish color, although it turns dark green over time and after sporulation; has several perpendicular ramifications, groups from 2 to 3 branches with pyramidal appearance; abundant conidia production, from 5 to 10 μm long and from 3.0 to 4.5 μm side. Grows in the laboratory on rice substrate with 47% humidity and at 28° C. for a 7-day period, in the presence of constant light.

(55) Stages Performed in the Lab for T. harzianum

(56) Procedures begin with the conditions in its natural habitat: Humidity: 30 to 40% and humidity is increased up to 70% and a Temperature from 28 to 30° C., maintained during all the research term. pH from 7.0 to 7.5. These conditions are the starting point to adapt the strain to thiodicarb and bifenthrin.

(57) To adapt this microorganism, 56 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 rate is at 82% and on stage E-2 at 85%. The remaining 83% 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 83 to 82%. At the end of stage E-4, organisms have been selected for 396 days.

(58) 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 83 to 82% (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 rate from 81 to 80%. At the end of E-8 (growth strengthening stage for the resistant strains) the adaptation process reaches 766 days.

(59) On stages E-9 and E-10, 0.3 LD (0.000053 gr) of thiodicarb are added, with a mortality rate from 78 to 76%. (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 of 75%. At the end of stage E-12 (growth strengthening stage for the resistant strains) the selection process reaches 1124 days.

(60) On stages E-13 and E-14, 0.3 LD (0.00000018 gr) of bifenthrin are added, with a mortality rate from 73 to 70% (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 rate of 69%. At the end of stage 16 (growth strengthening stage for the resistant strains) the selection process reaches 1368 days.

(61) On stages E-17 and E-18, 0.5 LD (0.000087 gr) of thiodicarb are added, with a mortality rate from 69 to 67% (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 of 66%. At the end of stage E-20 (growth strengthening stage for the resistant strains) the selection process reaches 1723 days.

(62) On stages E-21 and E-22, 0.5 LD (0.00000030 gr) of bifenthrin are added, with a mortality rate of 65% (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 65 to 63%. At the end of stage 24 (growth strengthening stage for the resistant strains) the selection process reaches 1975 days.

(63) On stages E-25 and E-26, 0.7 LD (0.00012 gr) of thiodicarb are added, with a mortality rate of 60% (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 58 to 56%. At the end of stage E-28 (growth strengthening stage for the resistant strains) the selection process reaches 2203 days.

(64) On stages E-29 and E-30, 0.7 LD (0.00000036 gr) of bifenthrin are added, with a mortality rate from 55 to 52% (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 48 to 44%. At the end of E-32 (growth strengthening stage for the resistant strains) the selection process reaches 23974 days.

(65) On stages E-33 and E-34, 0.9 LD (0.00016 gr) of thiodicarb are added, with a mortality rate from 42 to 40% (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 38 to 35%. At the end of stage E-36 (growth strengthening stage for the resistant strains) the selection process reaches 2556 days.

(66) On stages E-37 and E-38, 0.9 LD (0.00000042 gr) of bifenthrin are added, with a mortality rate from 24 to 22% (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 28 to 25%. At the end of stage 40 the selection process reaches 2686 days; 7.4 years of research.

(67) On stages E-41 and E-42, 1.1 LD (0.0002 gr) of thiodicarb are added, with a mortality rate from 24 to 22% (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 of 18%. At the end of stage E-28 (growth strengthening stage for the resistant strains) the selection process reaches 2805 days.

(68) On stages E-45 and E-46, 1.1 LD (0.00000054 gr) of bifenthrin are added, with a mortality rate from 20 to 18% (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 0.8 LD (0.00000060 gr) with a mortality rate of 14%. At the end of E-48 the selection process reaches 2914 days.

(69) On stages E-49 and E-50, 1.1 LD (0.00024 gr) of thiodicarb are added, with a mortality rate of 10% (The same way as in previous stages, E-50 is used to strengthen the growth of resistant strains). On stages E-51 and E-52, 1.2 LD (0.00026 gr) of thiodicarb are added, with a mortality rate of 8%. At the end of stage E-52 (growth strengthening stage for the resistant strains) the selection process reaches 2198 days.

(70) On stages E-53 and E-54, 1.2 LD (0.00000066 gr) of bifenthrin are added, with a mortality rate of 10% (The same way as in previous stages, E-54 is used to strengthen the growth of resistant strains). On stages E-55 and E-56, thiodicarb is increased to 1.3 LD (0.00000072 gr) with a mortality rate of 8%. At the end of E-56 (growth strengthening stage for the resistant strains) the selection process reaches 3001 days.

(71) TABLE-US-00003 TABLE NO. 3 Comparative Table, Initial and Final Characteristics DEVELOPMENT CHARACTERISTICS Trichoderma harzianum PARAMETER INITIAL FINAL pH 2 to 4.5 7 Humidity 60% 70% Respiration Aerobic Aerobic Temperature 24 to 27° C. 25 to 27° C. Resistant to: Organochlorinated, organic 1 LD of thiodicarb and phosphorus compounds and bifenthrin mercury Size 5 × 10 μm in diameter 6 × 10 μm in diameter Resistant to: N/A 1.3 LD of thiodicarb and bifenthrin
Metarhizium anisopliae

(72) This fungi is characterized by smooth hyphae, septated conidia predominantly cylindrical, grouped, green uninucleate with rounded endings, the average size was 6 mm long and 2.5 mm wide.

(73) The natural habitat for this fungi are ground larvae, commonly known as grub worms, scientific name Phillophaga, on sugar cane farmlands where this pest is present, causing significant damage to the crops.

(74) This fungi is characterized by smooth hyphae, septated conidia predominantly cylindrical, grouped, green uninucleate with rounded endings, the average size was 6 mm long and 2.5 mm wide.

(75) STAGES PERFORMED IN THE LAB FOR Metarhizium anisopliae

(76) Stages begin with the conditions in its natural habitat. It grows in the lab on rice substrate with 47% humidity for approximately 7 days at 28° C., and constant exposure to light on PDA for approximately 7 days at 28° C. 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.

(77) To adapt this microorganism, 40 stages were performed, beginning the process with the special growth media, which was enriched with minerals. 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 rate is rate is from 58 to 56%. The remaining 40% 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 of 55%. At the end of stage E-4, organisms have been selected for 220 days.

(78) 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 54 to 52% (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 rate from 50 to 48%. At the end of E-8 (growth strengthening stage for the resistant strains) the adaptation process reaches 421 days.

(79) On stages E-9 and E-10, 0.3 LD (0.000053 gr) of thiodicarb are added, with a mortality rate from 52 to 48%. (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 45 to 43%. At the end of stage E-12 (growth strengthening stage for the resistant strains) the selection process reaches 606 days.

(80) On stages E-13 and E-14, 0.3 LD (0.00000018 gr) of bifenthrin are added, with a mortality rate from 40 to 38% (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 rate from 38 to 35%. At the end of stage 16 (growth strengthening stage for the resistant strains) the selection process reaches 754 days.

(81) On stages E-17 and E-18, 0.5 LD (0.000087 gr) of thiodicarb are added, with a mortality rate from 34 to 33% (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 33 to 31%. At the end of stage E-20 (growth strengthening stage for the resistant strains) the selection process reaches 874 days.

(82) On stages E-21 and E-22, 0.5 LD (0.00000030 gr) of bifenthrin are added, with a mortality rate from 29 to 25% (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 22 to 20%. At the end of stage 24 (growth strengthening stage for the resistant strains) the selection process reaches 978 days.

(83) On stages E-25 and E-26, 0.7 LD (0.00012 gr) of thiodicarb are added, with a mortality rate from 18 to 15% (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 12 to 11%. At the end of stage E-28 (growth strengthening stage for the resistant strains) the selection process reaches 1060 days of adaptation.

(84) On stages E-29 and E-30, 0.7 LD (0.00000036 gr) of bifenthrin are added, with a mortality rate from 11 to 9% (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 7 to 5%. At the end of E-32 (growth strengthening stage for the resistant strains) the selection process reaches 1125 days.

(85) On stages E-33 and E-34, 0.9 LD (0.00016 gr) of thiodicarb are added, with a mortality rate of 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 of 15%. At the end of stage E-36 (growth strengthening stage for the resistant strains) the selection process reaches 2556 days of adaptation.

(86) On stages E-37 and E-38, 0.9 LD (0.00000042 gr) of bifenthrin are added, with a mortality rate from 24 to 22% (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 rate from 28 to 25%. At the end of stage 40 the selection process reaches 2686 days.

(87) TABLE-US-00004 TABLE NO. 4 Comparative Table, Initial and Final Characteristics DEVELOPMENT CHARACTERISTICS Metarhizium anisopliae PARAMETER INITIAL FINAL pH 2-4.5 7.0 Humidity 60% 60% Respiration Aerobic Aerobic Temperature 24-27° C. 25-27° C. Resistant to: N/A 1.3 LD of thiodicarb and bifenthrin

REFERENCES

(88) 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. U.S. Pat. No. 26,662 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 Apr. 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.

(89) 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 USB. List no. 51. International Journal of Systematic Bacteriology, 1994.

Invention Summary

(90) This invention provides the methodology to make a consortium of beneficial fungi resistant to carbamates contained in thiodicarb and the synthetic pyrethroid traces contained in bifenthrin.

(91) During the development of this research, performed in a time period over 5 years, the strains are subject to a physical media change, adapting them to withstand increasing concentrations of thiodicarb and bifenthrin.

(92) This compound enriches and increases the beneficial microorganism concentration, generating high-quality liquid biofertilizers, suitable to be used in agricultural production, in the soil conservation and recovery, complying with parameters established by sustainable organic agriculture where nature and the environment are maintained, recovered and used without causing negative impact.