Highly Concentrated Bacterial Liquid Soybean Inoculant
20210352910 · 2021-11-18
Inventors
Cpc classification
A01N25/04
HUMAN NECESSITIES
A01N63/20
HUMAN NECESSITIES
C12N1/04
CHEMISTRY; METALLURGY
A01N63/20
HUMAN NECESSITIES
International classification
A01N63/20
HUMAN NECESSITIES
C12N1/04
CHEMISTRY; METALLURGY
Abstract
This invention includes the composition of a highly concentrated liquid inoculant (between 10.sup.10 and 10.sup.11 cfu/mL) for soybean crops. The composition is packed and has a shelf life of up to 2 years stored at temperatures of up to 25° C. The inoculant may be applied in low doses onto seeds or in the furrow.
Claims
1. A highly concentrated bacterial liquid inoculant containing a concentration of bacteria per volume unit between 10.sup.10 and 10.sup.11 cfu/mL, with a shelf life of 2 years at storage temperatures up to 25° C., where the minimum concentration at expiry is 2×10.sup.10 cfu/mL.
2. The highly concentrated bacterial liquid inoculant in accordance with claim 1 whose bacteria are selected from the Bradyrhizobium japonicum, B. dizoefficiens or B. elkanii group, preferably Bradyrhizobium japonicum.
3. The highly concentrated bacterial liquid inoculant in accordance with claim 1, where the storage temperature may range between 2 to 25° C.
4. The highly concentrated bacterial liquid inoculant in accordance with claim 1, whose final formulation incorporates a stabilizer selected from: A1, A1810 and A2.
5. The highly concentrated bacterial liquid inoculant in accordance with claim 4, whose final formulation incorporates an A1 stabilizer.
6. The highly concentrated bacterial liquid inoculant in accordance with claim 1, where the inoculant may be applied onto the seed or in the sowing furrow.
7. The highly concentrated bacterial liquid inoculant in accordance with claim 1, where the application on the seed must be applied with at least one agrochemical product selected from: bacterial protector, nematicide, insecticide, fungicide, drying powder, nutritional supplement.
8. The highly concentrated bacterial liquid inoculant in accordance with claim 2, where the storage temperature may range between 2 to 25° C.
9. The highly concentrated bacterial liquid inoculant in accordance with claim 2, whose final formulation incorporates a stabilizer selected from: A1, A1810 and A2.
10. The highly concentrated bacterial liquid inoculant in accordance with claim 9, whose final formulation incorporates an A1 stabilizer.
11. The highly concentrated bacterial liquid inoculant in accordance with claim 2, where the inoculant may be applied onto the seed or in the sowing furrow.
12. The highly concentrated bacterial liquid inoculant in accordance with claim 3, where the inoculant may be applied onto the seed or in the sowing furrow.
13. The highly concentrated bacterial liquid inoculant in accordance with claim 4, where the inoculant may be applied onto the seed or in the sowing furrow.
14. The highly concentrated bacterial liquid inoculant in accordance with claim 2, where the application on the seed must be applied with at least one agrochemical product selected from: bacterial protector, nematicide, insecticide, fungicide, drying powder, nutritional supplement.
15. The highly concentrated bacterial liquid inoculant in accordance with claim 3, where the application on the seed must be applied with at least one agrochemical product selected from: bacterial protector, nematicide, insecticide, fungicide, drying powder, nutritional supplement.
16. The highly concentrated bacterial liquid inoculant in accordance with claim 4, where the application on the seed must be applied with at least one agrochemical product selected from: bacterial protector, nematicide, insecticide, fungicide, drying powder, nutritional supplement.
17. The highly concentrated bacterial liquid inoculant in accordance with claim 5, where the application on the seed must be applied with at least one agrochemical product selected from: bacterial protector, nematicide, insecticide, fungicide, drying powder, nutritional supplement.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0013]
[0014]
[0015]
[0016]
[0017]
[0018]
[0019]
[0020]
[0021]
DETAILED DESCRIPTION OF THE INVENTION AND DRAWINGS
[0022] Reference is made to the composition of a liquid inoculant highly concentrated in bacteria of the Bradyrhizobium genus that nodulate soybean plants, such as: B. japonicum; B. diazoefficiens; B. elkanii. The composition adapts to the usual commercial handling of inoculants under the usual storage and transport conditions, and it can reach a shelf life of up to two years and be applied at low doses.
[0023] Phase 1—Cellular Multiplication
[0024] Firstly, Bradyrhizobium bacteria, such as B. japonicum; B. diazoefficiens; B. elkanii, are multiplied in an appropriate culture medium such as the widely reported YMB (Yeast-Mannitol-Broth) or its derivations, such as the medium shown in Table 1, which is preferred for the composition of the highly concentrated inoculant to which this invention relates.
TABLE-US-00001 TABLE 1 Modified YMB medium - g/L Glycerol 25 Yeast Extract 4 KNO.sub.3 1.6 MgSO.sub.4•7H.sub.2O 0.2 K.sub.2HPO.sub.4 0.5 KH.sub.2PO.sub.4 0.5 (NH.sub.4).sub.2HPO.sub.4 0.4 NaCl 0.1 MnSO.sub.4•H.sub.2O 0.01 FeCl.sub.3 0.01 Water 1000 pH: 6.8-7.0
[0025] Bacteria are added to the culture medium, leaving an initial concentration of 1×10.sup.8 to 1×10.sup.9 cfu/mL, preferably 3×10.sup.8 to 7×10.sup.8 cfu/mL. Under appropriate temperature and agitation conditions, cellular multiplication suitable for this first stage of processing of the highly concentrated inoculant composition is obtained.
[0026] The appropriate incubation temperature ranges between 23 and 30° C., preferably between 27 and 30° C., and the degree of aeration is variable and depends on the element where the cellular multiplication takes place, ranging from Erlenmeyer flasks in an agitation incubator to bio-reactors or automated fermenters. Incubation time can range from 4 to 7 days depending on the bacterial strain considered within the species mentioned in this invention.
[0027] At the end of incubation, a level of bacterial concentration between 1.5×10.sup.10 to 3×10.sup.10 cfu/mL must be reached. This first stage of bacterial culture development is followed by Phase 2. which includes the process of centrifugation or cell separation from its surrounding medium, with the aim of achieving a highly concentrated bacterial broth ready to continue with the last phase (Phase 3) involving the final formulation process and packaging.
[0028] Phase 2—Cellular Concentration
[0029] The centrifugation or cell separation process concentrates the amount of bacteria in the bacterial broth from Phase 1. A laboratory centrifuge or an industrial separator can be used for this purpose. For the first case the proper centrifugation speed is between 5000 and 14000 rpm, and speeds between 8000 and 12000 rpm are preferred. Centrifugation time and temperature can vary between 5 and 20 minutes and between 18 and 26° C., respectively, preferably 8 to 15 minutes and 20 to 25° C.
[0030] Bacterial broth concentration means that from a normal bacterial broth a bacterial broth concentrated 8 to 20 times is obtained. To use the concentrated broth within the mentioned range, the sediment and part of the supernatant liquid must be used. The sum of the sediment volume and the supernatant volume represents the desired concentration within the mentioned range. The sediment is then vigorously mixed with the remaining supernatant and a homogeneous re-suspension of the highly concentrated bacterial broth is obtained.
[0031] This material is then ready for final formulation and packaging process (Phase 3), which will result in the highly concentrated inoculant.
[0032] Phase 3—Final Formulation
[0033] Once the 8× to 20× concentrated bacterial broth is obtained, the final formulation stabilizes the product to achieve a liquid composition with a shelf life of up to 2 years under storage conditions at room temperature of 20-25° C. The parameter defining the shelf life is the bacterial concentration ranging between 10.sup.10 and 10.sup.11 cfu/mL, more specifically between 2×10.sup.10 and 2×10.sup.11 cfu/mL, capable of producing optimal nodulation in the soybean crop.
[0034] The formulation consists of adding one or more substances mixed with water to the highly concentrated bacterial broth. The substances, dissolved in water, produce “stabilizing” options which can be added to the highly concentrated bacterial broth (Table 2). After the stabilizer is added to the broth, it is exposed to interaction under the same temperature and agitation conditions as in Phase 1.
[0035] The stabilizer used must be compatible with the viability of the bacteria and, at the same time, protect the cells from premature deterioration both in the container and on the seed or in the soil.
TABLE-US-00002 TABLE 2 Substance Substance concentration in Sucrose Trehalose CMC K.sub.2HPO.sub.4 KH.sub.2PO.sub.4 Water the Stabilizer (% Stabilizer (g) (g) (g) (g) (g) (mL) weight/weight) A1 2000 0 0 5 2.5 1000 66.7 A1810 1275 0 6.3 5 2.5 1000 56.3 A2 0 1000 0 5 2.5 1000 50.2
[0036] The stabilizer is incorporated proportionately ranging from 10 to 75% with respect to the volume of the highly concentrated bacterial broth. Preferably, the proportions used range between 20 and 50%.
[0037] Once the highly concentrated and stabilizing bacterial broth mixture is obtained, the formulation is subjected to an interaction time called “Process” (P) under the same temperature and agitation conditions as in Phase 1. Processes can vary between 0 and 5 days, preferably 2 to 4 days. The Process may increase the physicochemical assimilation of the stabilizer into the cells and may contribute to improve the stabilization and cell protection qualities of the highly concentrated liquid inoculant.
[0038] Once the formulation of the highly concentrated liquid inoculant is complete, it is packaged. The composition can be placed in single- or double-layer low density polyethylene bladders, each layer 35 to 80 μm thick, preferably 50 to 70 μm, using a volume of 0.5 to 15 L, preferably 0.5 to 2 L. This type of packaging allows the gaseous exchange between the composition and the environment, contributing to the metabolic balance of the bacteria and their stabilization during the product's life. In addition, it maintains purity and prevents contamination of particles inside.
[0039] The packaged product can be stored at temperatures between 2 to 25° C., although the composition design allows temperatures between 20 to 25° C. while maintaining its useful life for up to 2 years. Given the type of packaging used, no specific relative air humidity is required. There are no specific requirements for brightness either, although it should be preferably stored in the dark.
EXAMPLES
Example 1
[0040] Survival of Bradyrhizobium japonicum on Soybean and Nodulation Response in Treatments with Agrochemicals Used in Brazil
[0041] The following example shows the results of the use of the highly concentrated inoculant after storage for 1.5 years from its manufacture. The effect of two preservation temperatures of the treated seed was evaluated (20-24° C. and 26° C.). In addition, it was applied with different commercial agrochemicals commonly used in Brazil and its performance was evaluated. The highly concentrated inoculant was applied in 2 different doses and using different application methods. The results were evaluated: the viability of the bacteria on the soybean was measured, the number of bacteria per seed were counted, and the nodulation response in the plant growth chamber was observed, according to the time since the treatment (pre-inoculation).
[0042] The highly concentrated liquid inoculant used in this example was manufactured from Stabilizer A1 and contained 1.65×10.sup.11 cfu/mL, with 1.5 year since its manufacture. Prior to application on soybeans, the inoculant (UHC) was mixed with a bacterial protector (Premax LLI). The mixture was let sit for 15 minutes and the proper dose was applied to 200 g of DM 4014 soybean which had been previously treated with commercial agrochemicals. It was also considered a control without agrochemicals. Table 3 shows the products that were used and their application rates on the seeds.
TABLE-US-00003 TABLE 3 Dosage (quantity/kg Product name Product type Active ingredient (%) soybean seed) UHC Highly Bradyrhizobium japonicum + 3 mL (dose1) concentrated Bradyrhizobium diazoefficiens (15) or 4 mL bacterial (dose2) inoculant Premax LLI Bacterial Sugar (% not applicable) 1 mL protector Avicta 500 FS Nematicide and Abamectine (50) 1 mL insecticide Cruiser 35 FS Insecticide Thiamethoxam (35) 2 mL Maxim Fungicide Tiabendazole (15) + 1 mL Evolution fludioxonil (2.5) + metalaxyl-M (2) Fluidus 028 Drying powder Micas (% not applicable) 1 g Amulet Insecticide Fipronil (20) 1 mL Cropstar Insecticide Imidacloprid (15) + thiodicarb (45) 6 mL Ritiram Carb Fungicide Thiram (35) + carbendazim (15) 2 mL Plus Standak Top Fungicide and Pyraclostrobin (2.5) + Methyl 2 mL insecticide thiophanate (22.5) + Fipronil (25) Dermacor Insecticide Chlorantraniliprole (62.5) 1 mL Seed + Dry Nutrition Ca (4.2) + Mg (1.9) + S (4.2) + Co 2.5 g supplement (0.0012) + Cu (0.08) + Fe (0.9) + Mn (0.25) + Mo (0.0008) + Zn (1)
[0043] Levels 34, 63 and 78 days after inoculation (dai) were established, which are the times between seed treatment and the biological determinations (early seed treatment or pre-inoculation). The treated seed was stored at two temperatures (20-24° C. and 26° C.) and in darkness.
[0044] Technics and Measurements of Results
[0045] The number of viable Bradyrhizobium per seed was determined using the technique of decimal dilutions and surface dissemination (0.1 mL of inoculum) in Petri dishes containing YMA (Yeast-Mannitol-Agar) medium. In each determination, 50 seeds were shaken for 12 min with 50 mL of purified (demineralized) water sterilized by autoclave, in a 125 mL bottle, at 200 rpm. Three repetitions and 2 dishes by dilution were used. The sown dishes were incubated at 29.5° C. and Bradyrhizobium colonies were counted 7 days later.
[0046] For the nodulation study, seed was sown in cups with 330 g of sandy soil free of Bradyrhizobium (from La Pampa, Argentina, with no soybean crop history). The units were placed in a growth chamber under controlled conditions of temperature (25-28° C.), soil moisture (close to field capacity) and air (60-70% RH), light (8000 lux) and photoperiod (16 h light, 8 h darkness). Irrigation was carried out with purified (demineralized) water sterilized by filtration (0.1 μm). Two seeds per glass were sown and on the fourth day a thinning of 1 plant per glass was done. We worked with 9 repetitions and 35 days after sowing the plants were processed to determine the 4 nodulation variables: number of nodules per plant (by direct count) in primary and total root; and dry mass of nodules per plant (by weighing nodules dried at 60° C. for 48 hours) in primary and total root.
[0047] The response of the treatments can be evaluated according to benchmarks as suitability or agronomic efficiency indicators. One of these benchmarks may be one that meets the minimum acceptable values of two or more of the variables analyzed: Bradyrhizobium cfu/seed, ≥5×10.sup.4; Number of nodules per plant in primary root ≥2; Number of nodules per plant in total root ≥3; dry mass (mg) of nodules per plant in primary root ≥10; and dry mass (mg) of nodules per plant in total root ≥15. In order to determine suitability or efficiency, the following variables are preferably taken into account: number of nodules per plant in total root and dry mass (mg) of nodules per plant in total root.
[0048] Results
[0049] Table 4 shows that the highly concentrated (UHC) bacterial inoculant was efficient, as it responded to several cases with ≥3 nodules per plant in total root and ≥15 mg of dry mass of nodules in total root. Suitability was influenced by the degree of compatibility with agrochemicals, seed storage temperature, pre-inoculation time and inoculant dose. A suitable response was achieved of up to 78 days after inoculation (dai) in the presence of different combinations of agrochemicals and seed storage at 20-24° C. The best response under these conditions was observed in seed treatments with “Maxim Evolution+Standak Top//UHC+Premax LLI dose1”, “Avicta 500 FS+Cruiser 35 FS+Maxim Evolution//UHC+Premax LLI dose2” and “UHC+Premax LLI dose2/Fluidus 028”, which produced ≥6 nodules per plant in total root and ≥32.3 mg of dry mass of nodules in total root.
TABLE-US-00004 TABLE 4 Dry Dry mass mass No of No of (mg) (mg) Seed nodules/ nodules/ nodules/ nodules/ storage plant in plant in plant in plant in temperature Seed cfu/ Primary Total Primary Total dai (° C.) treatment seed Root Root Root Root 34 20-24 UHC + Premax LLI 5.72E+05 4.6 6.9 48.3 60.0 dose1 34 20-24 Avicta 500 FS + 3.08E+05 4.4 18.5 35.9 67.1 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose1 34 20-24 Avicta 500 FS + 1.97E+05 1.6 11.3 19.6 56.6 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose1/Fluidus 028 34 20-24 Amulet + Cruiser 35 1.15E+05 3.6 13.1 33.2 63.3 FS + Maxim Evolution // UHC + Premax LLI dose1 34 20-24 Cropstar // UHC + 4.75E+03 0.0 0.9 0.0 5.0 Premax LLI dose1 34 20-24 Maxim Evolution + 2.77E+05 0.0 3.3 0.0 17.8 Ritiram Carb Plus // UHC + Premax LLI dose1 34 20-24 Maxim Evolution + 1.95E+05 0.0 3.3 0.0 13.8 Ritiram Carb Plus // UHC + Premax LLI dose1/Fluidus 028 34 20-24 Maxim Evolution + 1.04E+06 1.4 15.6 9.8 53.4 Standak Top // UHC + Premax LLI dose1 34 20-24 Dermacor // UHC + 7.77E+05 2.1 14.1 19.9 49.0 Premax LLI dose1 34 20-24 UHC + Premax LLI 4.57E+05 1.0 14.7 10.4 49.4 dose1/Fluidus 028 34 20-24 Standak Top // UHC + 3.90E+05 2.9 9.0 27.2 48.9 Premax LLI dose1 34 20-24 UHC + Premax LLI 9.17E+04 0.8 4.2 6.8 28.3 dose1/Seed + Dry 34 20-24 UHC + Premax LLI 1.79E+06 2.3 15.9 30.6 55.0 dose2 34 20-24 Avicta 500 FS + 1.05E+06 2.7 12.1 29.2 63.2 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose2 34 20-24 Avicta 500 FS + 6.68E+05 3.8 11.2 26.9 55.3 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose2/Fluidus 028 34 20-24 Amulet + Cruiser 35 7.50E+05 4.2 15.2 37.0 71.1 FS + Maxim Evolution // UHC + Premax LLI dose2 34 20-24 Cropstar // UHC + 9.00E+03 0.0 1.0 0.0 10.2 Premax LLI dose2 34 20-24 Maxim Evolution + 1.04E+06 0.0 3.2 0.0 22.4 Ritiram Carb Plus // UHC + Premax LLI dose2 34 20-24 Maxim Evolution + 2.42E+05 0.0 3.8 0.0 23.8 Ritiram Carb Plus // UHC + Premax LLI dose2/Fluidus 028 34 20-24 Maxim Evolution + 1.14E+06 1.1 17.7 11.7 60.9 Standak Top // UHC + Premax LLI dose2 34 20-24 Dermacor // UHC + 1.57E+06 4.2 7.8 48.4 57.9 Premax LLI dose2 34 20-24 UHC + Premax LLI 9.87E+05 2.2 10.2 25.2 54.8 dose2/Fluidus 028 34 20-24 Standak Top // UHC + 6.18E+05 6.4 13.8 48.0 65.2 Premax LLI dose2 34 20-24 UHC + Premax LLI 1.20E+05 0.2 5.2 5.2 26.7 dose2/Seed + Dry 34 26 UHC + Premax LLI 3.17E+05 3.2 13.2 39.4 54.2 dose1 34 26 Avicta 500 FS + 2.67E+05 1.8 13.9 17.4 57.6 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose1 34 26 Avicta 500 FS + 7.60E+04 0.8 6.2 7.0 38.0 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose1/Fluidus 028 34 26 Amulet + Cruiser 35 1.15E+05 0.0 6.9 0.0 46.9 FS + Maxim Evolution // UHC + Premax LLI dose1 34 26 Cropstar // UHC + 8.33E+03 0.0 1.0 0.0 8.1 Premax LLI dose1 34 26 Maxim Evolution + 8.17E+04 0.0 3.2 0.0 10.8 Ritiram Carb Plus // UHC + Premax LLI dose1 34 26 Maxim Evolution + 1.38E+05 0.0 0.8 0.0 4.7 Ritiram Carb Plus // UHC + Premax LLI dose1/Fluidus 028 34 26 Maxim Evolution + 1.50E+05 0.1 10.1 0.7 49.6 Standak Top // UHC + Premax LLI dose1 34 26 Dermacor // UHC + 1.28E+05 0.4 7.6 7.3 36.7 Premax LLI dose1 34 26 UHC + Premax LLI 1 24E+05 0.6 10.9 6.8 50.8 dose1/Fluidus 028 34 26 Standak Top // UHC + 8.27E+04 0.0 7.9 0.0 49.2 Premax LLI dose1 34 26 UHC + Premax LLI 2.08E+04 0.0 0.9 0.0 10.2 dose1/Seed + Dry 34 26 UHC + Premax LLI 7.28E+05 2.7 6.9 38.3 52.3 dose2 34 26 Avicta 500 FS + 1.43E+05 0.1 8.8 1.1 49.8 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose2 34 26 Avicta 500 FS + 1.07E+05 0.1 5.8 1.8 41.2 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose2/Fluidus 028 34 26 Amulet + Cruiser 35 1.02E+05 0.3 10.6 6.2 46.7 FS + Maxim Evolution // UHC + Premax LLI dose2 34 26 Cropstar // UHC + 6.00E+03 0.0 0.1 0.0 1.1 Premax LLI dose2 34 26 Maxim Evolution + 5.38E+04 0.0 0.6 0.0 7.1 Ritiram Carb Plus // UHC + Premax LLI dose2 34 26 Maxim Evolution + 5.40E+04 0.0 0.4 0.0 4.9 Ritiram Carb Plus // UHC + Premax LLI dose2/Fluidus 028 34 26 Maxim Evolution + 1.91E+05 0.3 13.9 3.8 59.2 Standak Top // UHC + Premax LLI dose2 34 26 Dermacor // UHC + 1.88E+05 1.9 9.0 18.3 45.6 Premax LLI dose2 34 26 UHC + Premax LLI 3.48E+05 1.3 6.7 20.4 48.2 dose2/Fluidus 028 34 26 Standak Top // UHC + 1.17E+05 1.1 9.1 11.3 56.0 Premax LLI dose2 34 26 UHC + Premax LLI 4.33E+04 0.2 2.9 3.4 16.4 dose2/Seed + Dry 63 20-24 UHC + Premax LLI 7.58E+04 1.4 10.3 18.6 35.1 dose1 63 20-24 Avicta 500 FS + 3.72E+04 0.0 11.0 0.0 51.2 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose1 63 20-24 Avicta 500 FS + 2.40E+04 0.3 5.4 6.5 40.0 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose1/Fluidus 028 63 20-24 Amulet + Cruiser 35 8.50E+03 0.0 7.3 0.0 40.3 FS + Maxim Evolution // UHC + Premax LLI dose1 63 20-24 Cropstar // UHC + 3.65E+04 0.0 0.2 0.0 1.0 Premax LLI dose1 63 20-24 Maxim Evolution + 1.50E+04 0.0 0.4 0.0 2.1 Ritiram Carb Plus // UHC + Premax LLI dose1 63 20-24 Maxim Evolution + 3.83E+03 0.0 0.4 0.0 1.7 Ritiram Carb Plus // UHC + Premax LLI dose1/Fluidus 028 63 20-24 Maxim Evolution + 3.17E+04 0.1 5.7 3.0 40.3 Standak Top // UHC + Premax LLI dose1 63 20-24 Dermacor // UHC + 4.25E+04 0.3 4.4 8.6 23.8 Premax LLI dose1 63 20-24 UHC + Premax LLI 6.50E+04 1.0 5.8 15.7 42.3 dose1/Fluidus 028 63 20-24 Standak Top // UHC + 1.00E+04 0.0 4.4 0.0 30.6 Premax LLI dose1 63 20-24 UHC + Premax LLI 1.50E+04 0.0 2.1 0.0 14.7 dose1/Seed + Dry 63 20-24 UHC + Premax LLI 1.47E+05 1.8 7.0 23.9 48.8 dose2 63 20-24 Avicta 500 FS + 7.95E+04 0.0 7.0 0.0 50.3 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose2 63 20-24 Avicta 500 FS + 6.83E+04 0.0 2.9 0.0 18.2 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose2/Fluidus 028 63 20-24 Amulet + Cruiser 35 3.83E+04 0.3 6.0 5.3 43.7 FS + Maxim Evolution // UHC + Premax LLI dose2 63 20-24 Cropstar // UHC + 2.00E+03 0.0 0.3 0.0 2.1 Premax LLI dose2 63 20-24 Maxim Evolution + 2.67E+04 0.0 0.0 0.0 0.0 Ritiram Carb Plus // UHC + Premax LLI dose2 63 20-24 Maxim Evolution + 7.50E+03 0.9 4.9 10.6 39.4 Ritiram Carb Plus // UHC + Premax LLI dose2/Fluidus 028 63 20-24 Maxim Evolution + 2.00E+04 0.2 3.6 3.4 23.7 Standak Top // UHC + Premax LLI dose2 63 20-24 Dermacor // UHC + 4.50E+04 0.0 4.8 0.0 30.0 Premax LLI dose2 63 20-24 UHC + Premax LLI 1.05E+05 0.8 2.7 11.8 24.2 dose2/Fluidus 028 63 20-24 Standak Top // UHC + 4.50E+03 0.2 6.6 3.2 43.2 Premax LLI dose2 63 20-24 UHC + Premax LLI 2.33E+04 0.0 0.7 0.0 1.8 dose2/Seed + Dry 63 26 UHC + Premax LLI 1.65E+04 0.2 4.9 4.1 32.6 dose1 63 26 Avicta 500 FS + 3.50E+03 0.0 4.3 0.0 26.1 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose1 63 26 Avicta 500 FS + 1.33E+03 0.0 1.4 0.0 9.2 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose1/Fluidus 028 63 26 Amulet + Cruiser 35 2.17E+03 0.0 2.6 0.0 18.1 FS + Maxim Evolution // UHC + Premax LLI dose1 63 26 Cropstar // UHC + 1.00E+03 0.0 0.0 0.0 0.0 Premax LLI dose1 63 26 Maxim Evolution + 2.25E+03 0.0 0.0 0.0 0.0 Ritiram Carb Plus // UHC + Premax LLI dose1 63 26 Maxim Evolution + 1.00E+03 0.0 0.4 0.0 0.3 Ritiram Carb Plus // UHC + Premax LLI dose1/Fluidus 028 63 26 Maxim Evolution + 2.00E+03 0.0 0.9 0.0 4.9 Standak Top // UHC + Premax LLI dose1 63 26 Dermacor // UHC + 1.75E+03 0.0 1.3 0.0 9.4 Premax LLI dose1 63 26 UHC + Premax LLI 1.33E+04 0.0 4.0 0.0 17.4 dose1/Fluidus 028 63 26 Standak Top // UHC + 1.00E+03 0.0 1.4 0.0 22.6 Premax LLI dose1 63 26 UHC + Premax LLI 2.25E+03 0.0 0.4 0.0 2.1 dose1/Seed + Dry 63 26 UHC + Premax LLI 2.17E+04 1.1 6.1 23.6 45.0 dose2 63 26 Avicta 500 FS + 4.00E+03 0.1 2.2 1.9 21.7 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose2 63 26 Avicta 500 FS + 2.67E+03 0.0 1.2 0.0 8.4 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose2/Fluidus 028 63 26 Amulet + Cruiser 35 3.50E+03 0.0 1.7 0.0 10.4 FS + Maxim Evolution // UHC + Premax LLI dose2 63 26 Cropstar // UHC + 1.00E+03 0.0 0.1 0.0 1.1 Premax LLI dose2 63 26 Maxim Evolution + 1.00E+03 0.0 0.3 0.0 2.0 Ritiram Carb Plus // UHC + Premax LLI dose2 63 26 Maxim Evolution + 1.75E+03 0.0 0.0 0.0 0.0 Ritiram Carb Plus // UHC + Premax LLI dose2/Fluidus 028 63 26 Maxim Evolution + <1.00E+03 0.0 2.3 0.0 14.3 Standak Top // UHC + Premax LLI dose2 63 26 Dermacor // UHC + 2.50E+03 0.0 0.7 0.0 11.6 Premax LLI dose2 63 26 UHC + Premax LLI 4.28E+04 0.0 3.8 0.0 21.6 dose2/Fluidus 028 63 26 Standak Top // UHC + 1.80E+04 0.0 2.4 0.0 19.4 Premax LLI dose2 63 26 UHC + Premax LLI 1.35E+04 0.0 0.8 0.0 5.1 dose2/Seed + Dry 78 20-24 UHC + Premax LLI 1.62E+04 0.0 6.0 0.0 27.1 dose1 78 20-24 Avicta 500 FS + 1.03E+04 0.0 3.9 0.0 28.1 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose1 78 20-24 Avicta 500 FS + 4.33E+03 0.0 0.4 0.0 6.3 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose1/Fluidus 028 78 20-24 Amulet + Cruiser 35 5.67E+03 0.1 1.1 1.6 13.4 FS + Maxim Evolution // UHC + Premax LLI dose1 78 20-24 Cropstar // UHC + <1.00E+03 0.0 0.4 0.0 2.9 Premax LLI dose1 78 20-24 Maxim Evolution + 2.00E+03 0.0 0.5 0.0 3.8 Ritiram Carb Plus // UHC + Premax LLI dose1 78 20-24 Maxim Evolution + 2.17E+03 0.0 0.8 0.0 3.0 Ritiram Carb Plus // UHC + Premax LLI dose1/Fluidus 028 78 20-24 Maxim Evolution + 6.00E+03 2.4 6.6 19.6 32.3 Standak Top // UHC + Premax LLI dose1 78 20-24 Dermacor // UHC + 5.67E+03 1.4 3.3 4.4 8.4 Premax LLI dose1 78 20-24 UHC + Premax LLI 1.73E+04 2.0 6.3 14.1 28.3 dose1/Fluidus 028 78 20-24 Standak Top // UHC + 1.38E+04 1.4 5.4 3.9 16.4 Premax LLI dose1 78 20-24 UHC + Premax LLI 2.83E+03 0.0 0.6 0.0 1.1 dose1/Seed + Dry 78 20-24 UHC + Premax LLI 5.40E+04 2.4 8.4 23.5 41.8 dose2 78 20-24 Avicta 500 FS + 1.50E+04 1.0 6.0 7.6 32.8 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose2 78 20-24 Avicta 500 FS + 7.83E+03 1.5 5.3 9.5 23.9 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose2/Fluidus 028 78 20-24 Amulet + Cruiser 35 1.25E+04 1.5 3.5 15.6 24.6 FS + Maxim Evolution // UHC + Premax LLI dose2 78 20-24 Cropstar // UHC + 1.00E+03 0.1 0.8 6.0 9.4 Premax LLI dose2 78 20-24 Maxim Evolution + 3.67E+03 0.1 0.3 0.5 0.6 Ritiram Carb Plus // UHC + Premax LLI dose2 78 20-24 Maxim Evolution + 7.50E+03 0.1 0.8 0.1 1.5 Ritiram Carb Plus // UHC + Premax LLI dose2/Fluidus 028 78 20-24 Maxim Evolution + 5.67E+03 0.8 3.1 4.5 14.1 Standak Top // UHC + Premax LLI dose2 78 20-24 Dermacor // UHC + 8.17E+03 0.3 2.9 7.1 13.6 Premax LLI dose2 78 20-24 UHC + Premax LLI 2.17E+04 2.0 6.1 31.0 39.4 dose2/Fluidus 028 78 20-24 Standak Top // UHC + 7.50E+03 0.4 3.3 5.6 22.6 Premax LLI dose2 78 20-24 UHC + Premax LLI 3.50E+03 0.1 1.6 0.1 4.8 dose2/Seed + Dry 78 26 UHC + Premax LLI 4.93E+03 0.0 1.4 0.0 7.4 dose1 78 26 Avicta 500 FS + 3.17E+02 0.0 1.8 0.0 12.4 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose1 78 26 Avicta 500 FS + 3.00E+02 0.0 0.1 0.0 0.8 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose1/Fluidus 028 78 26 Amulet + Cruiser 35 1.83E+02 0.0 0.3 0.0 5.5 FS + Maxim Evolution // UHC + Premax LLI dose1 78 26 Cropstar // UHC + <1.00E+02 0.0 0.1 0.0 0.8 Premax LLI dose1 78 26 Maxim Evolution + 2.00E+02 0.0 0.0 0.0 0.0 Ritiram Carb Plus // UHC + Premax LLI dose1 78 26 Maxim Evolution + 1.05E+03 0.0 0.5 0.0 0.4 Ritiram Carb Plus // UHC + Premax LLI dose1/Fluidus 028 78 26 Maxim Evolution + 2.25E+02 0.0 0.8 0.0 3.9 Standak Top // UHC + Premax LLI dose1 78 26 Dermacor // UHC + 1.95E+03 0.0 0.1 0.0 0.5 Premax LLI dose1 78 26 UHC + Premax LLI 5.67E+02 0.3 1.1 1.8 3.8 dose1/Fluidus 028 78 26 Standak Top // UHC + 3.83E+02 0.4 0.9 3.4 4.9 Premax LLI dose1 78 26 UHC + Premax LLI 2.17E+02 0.0 0.1 0.0 0.1 dose1/Seed + Dry 78 26 UHC + Premax LLI 2.78E+03 0.8 2.0 2.9 10.9 dose2 78 26 Avicta 500 FS + 1.50E+03 0.8 2.3 11.5 18.3 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose2 78 26 Avicta 500 FS + 3.50E+02 0.1 1.8 0.6 5.4 Cruiser 35 FS + Maxim Evolution // UHC + Premax LLI dose2/Fluidus 028 78 26 Amulet + Cruiser 35 3.00E+02 0.0 0.9 0.0 3.5 FS + Maxim Evolution // UHC + Premax LLI dose2 78 26 Cropstar // UHC + 1.00E+02 0.0 0.5 0.0 1.9 Premax LLI dose2 78 26 Maxim Evolution + 1.00E+02 0.0 0.3 0.0 0.1 Ritiram Carb Plus // UHC + Premax LLI dose2 78 26 Maxim Evolution + 2.00E+02 0.0 0.8 0.0 1.8 Ritiram Carb Plus // UHC + Premax LLI dose2/Fluidus 028 78 26 Maxim Evolution + 3.00E+02 0.0 0.4 0.0 1.8 Standak Top // UHC + Premax LLI dose2 78 26 Dermacor // UHC + 1.00E+02 0.0 0.0 0.0 0.0 Premax LLI dose2 78 26 UHC + Premax LLI 1.25E+03 0.0 0.3 0.0 0.9 dose2/Fluidus 028 78 26 Standak Top // UHC + <1.00E+02 0.4 1.0 5.4 11.0 Premax LLI dose2 78 26 UHC + Premax LLI 2.67E+03 0.0 0.1 0.0 0.1 dose2/Seed + Dry dai = days after inoculation (pre-inoculation). “/” = wet sequential method (the first products are applied and then the remaining products are applied onto the wet seed); “//” = dry sequential method (the first products are applied and after several minutes the remaining products are applied onto the dry seed); “+” = simultaneous method (the products are mixed and after a few minutes the mixture is applied onto the seed). UHC + Premax LLI dose1 = 3 mL highly concentrated Inoculant/kg + 1 mL Premax LLI/kg; UHC + Premax LLI dose2 = 4 mL highly concentrated Inoculant/kg + 1 mL Premax LLI/kg.
Example 2
[0050] Survival of Bradyrhizobium japonicum on Soybean Seed and Nodulation Response in Treatments with Agrochemicals Used in the United States and Canada
[0051] This example used the highly concentrated inoculant after 2 years storage since manufacture. In addition, its performance was evaluated in comparison with different commercial agrochemicals commonly used in the United States or Canada. The highly concentrated inoculant was applied in 5 different doses and using different application methods. The results were evaluated according to the viability of the bacteria on the soybean, the number of bacteria per seed; the nodulation response in the plant growth chamber, and the time since treatment (pre-inoculation).
[0052] The highly concentrated liquid inoculant used in this example was manufactured from Stabilizer A1 and contained 2.15×10.sup.11 cfu/mL, with 2 years since its manufacture. In some seed treatments, the right dose of inoculant (UHC) was directly applied to the seed and in other cases, the inoculant was previously mixed with a bacterial protector (Premax); the mix was let sit for 15 minutes and the proper dose was applied onto the seed. Each seed treatment involved 400 g of DM 3312 variety soybean, recently treated with commercial agrochemicals in widespread use in the United States and/or Canadian markets. Non-agrochemical controls were also considered. Table 5 shows the products used and their application rates on the seeds.
TABLE-US-00005 TABLE 5 Dosage (quantity/kg Product name Product type Active ingredient (%) soybean seed) UHC Highly Bradyrhizobium japonicum + 0.98 mL, 0.73 mL, concentrated Bradyrhizobium diazoefficiens 0.65 mL, 0.5 mL bacterial (15) or 0.48 mL inoculant Premax Bacterial Sugar (% not applicable) 0.5 mL, 0.25 mL protector or 0.15 mL Intego Suite Fungicide Clothianidin (20.06) + ethaboxam 2.2 mL Soybeans (2.97) + ipconazole (0.99) + metalaxyl (0.79) CruiserMaxx Insecticide Thiamethoxam (20.8) + 2.11 mL Vibrance and fungicide mefenoxam (3.13) + fludioxonil (1.04) + sedaxane (1.04)
[0053] Levels 34, 77, 120, 149, 181 and 210 days after inoculation (dai) were established, corresponding to the time between the seed treatment and the biological determinations (early seed treatment or pre-inoculation). Storage of the treated seed was in the temperature conditions closest to those in the U.S. and Canadian warehouses, starting with the equivalent of seed treatment in November (fall) (Table 6).
TABLE-US-00006 TABLE 6 Storage temperature Storage time of treated of treated seed (° C.) seed (dai) 5 0 to 30 2 31 to 120 5 121 to 150 10 151 to 180 16 181 to 210
[0054] The test results were measured in the same way as in the previous example, extensively detailed in the section “Technics and Measurement of Results”. The test results can be seen graphically in