USE OF CYAMOPSIS TETRAGONOLOBA (GUAR) GUM FOR MICROORGANISMS GROWTH

Abstract

The present invention relates to the in vitro use of Cyamopsis tetragonoloba (guar) gum for maintaining or increasing the growth rate of microorganisms.

Claims

1. A method, comprising maintaining or increasing the growth rate of microorganisms by using Cyamopsis tetragonoloba (guar) gum in vitro, or on a plant, on a seed or in soil.

2. (canceled)

3. The method of claim 1, wherein the microorganisms are fungi or bacteria.

4. The method of claim 1, wherein the growth rate of microorganisms is increased.

5. The method of claim 1, wherein the microorganisms are selected from the group consisting of Gram-positive bacteria.

6. The method of claim 1, wherein the microorganisms are selected from the group consisting of Gram-negative bacteria.

7. The method of claim 1, wherein the microorganism and the Cyamopsis tetragonoloba (guar) gum are combined in a ratio microorganism:Cyamopsis tetragonoloba (guar) gum ranging from 1.Math.10.sup.4 to 1.Math.10.sup.15 CFU/g.

8. A method for maintaining or increasing the growth rate of microorganisms, the method comprising a step of contacting at least one seed with Cyamopsis tetragonoloba (guar) gum.

9. (canceled)

10. (canceled)

11. A biostimulant composition comprising at least one microorganism and at least Cyamopsis tetragonoloba (guar) gum.

12. The biostimulant composition of claim 11, wherein the microorganism and the Cyamopsis tetragonoloba (guar) gum are combined in a ratio microorganism:Cyamopsis tetragonoloba (guar) gum ranging from 1.Math.10.sup.4 to 1.Math.10.sup.15 CFU/g.

13. A kit comprising at least one microorganism and at least Cyamopsis tetragonoloba (guar) gum.

14. (canceled)

15. A seed coated with the biostimulant composition of claim 11.

16. The method of claim 7, wherein the microorganism and the Cyamopsis tetragonoloba (guar) gum are combined in a ratio microorganism:Cyamopsis tetragonoloba (guar) gum ranging from 1.Math.10.sup.4 to 1.Math.10.sup.12 CFU/g.

17. The method of claim 16, wherein the microorganism and the Cyamopsis tetragonoloba (guar) gum are combined in a ratio microorganism:Cyamopsis tetragonoloba (guar) gum ranging from 1.Math.10.sup.4 to 1.Math.10.sup.11 CFU/g.

18. The method of claim 17, wherein the microorganism and the Cyamopsis tetragonoloba (guar) gum are combined in a ratio microorganism:Cyamopsis tetragonoloba (guar) gum ranging from 1.Math.10.sup.4 to 5.Math.10.sup.10 CFU/g.

19. The method of claim 18, wherein the microorganism and the Cyamopsis tetragonoloba (guar) gum are combined in a ratio microorganism:Cyamopsis tetragonoloba (guar) gum ranging from 1.Math.10.sup.5 to 1.Math.10.sup.10 CFU/g.

20. The method of claim 8, wherein the microorganisms are bacteria.

21. The biostimulant composition of claim 11, wherein the at least one microorganism is a bacterium.

Description

EXAMPLES

Example 1

[0092] The following materials are used in the experiments:

[0093] Guar: Cyamopsis tetragonoloba (guar) gum, available from Solvay (provided as a powder)

[0094] Bacteria strains were acquired from Tropical Culture Collection in André Tosello Foundation—Brazil. [0095] Bacillus subtilis CCT 0089 [0096] Bacillus megaterium CCT 0536 [0097] Agrobacterium radiobacter CCT 4774 [0098] Bradyrhizobium japonicum CCT 4065

[0099] All strains were stored at −80° C. in the appropriated culture media, containing 15% of glycerol.

[0100] Two different culture media were used in the experiments: [0101] NA media containing per liter: 3 g of meat extract, 5 g of peptone and 15 g of agar (for solid media only) [0102] YMA media containing per liter: 0.5 g of monobasic potassium phosphate, 0.2 g of magnesium sulphate; 0.1 g of sodium chloride; 0.5 g of yeast extract; 10 g of mannitol (for inoculum and solid media only); 5 mL of a 5% bromothymol blue solution and 15 g of agar (solid media only).

[0103] For strains Bacillus subtilis, Bacillus megaterium and Agrobacterium radiobacter NA media was used. For strain Bradyrhyzobium japonicum, YMA media was used. These media were selected according to strains supplier.

[0104] A 250 mL shake flask containing 100 mL of NA or YMA culture media, was inoculated with 1 mL of the stock culture and incubated at 30° C., 150 rpm for 72 hours.

[0105] For each strain, 10 mL of the reactivation media were then transferred into a 250 mL shake flask containing 100 mL of the same media, with the addition of guar powder (at 0.7 wt % in the incubation media); and incubated at 30° C., 150 rpm, for 96 hours. An experiment without addition of guar powder is also performed for each strain as a control.

[0106] 1004 samples of each experiments were taken after 0 h, 24 h, 48 h, 72 h and 96 h of incubation. These samples were diluted (the dilutions were variable according to strain growth, being from 1×10.sup.−5 up to 1×10.sup.−15) and the dilutions plated in solid NA or YMA media. The plates were incubated at 30° C. until appearance of colonies. After incubation, the number of colonies present in each dilution was counted and used to evaluate bacterial growth.

[0107] For bacterial growth rate determination, a graph of the log.sub.10(number of colonies) versus time of incubation was constructed. The straight line in this graph represents the exponential phase of bacterial growth and the angular coefficient represents the bacterial growth rate (μ).

[0108] The μ value was used to compare all the experiments and to evaluate the influence of guar addition on bacterial growth.

Example 1a

[0109] In a first set of experiments the ratio of microorganisms and guar is equal to 1.Math.00×10.sup.6 CFU/g. The bacteria growth rate (μ) obtained for the different experiments are summarized in Table 1a:

TABLE-US-00001 TABLE 1a Bacteria growth Composition rate (h.sup.−1) Bacillus subtilis CCT 0089 0.0647 Bacillus subtilis CCT 0089 + guar 0.0657 Bacillus megaterium CCT 0536 0.0605 Bacillus megaterium CCT 0536 + guar 0.0878 Agrobacterium radiobacter CCT 4774 + guar 0.0509 Agrobacterium radiobacter CCT 4774 0.0681 Bradyrhyzobium japonicum CCT 4065 0.0891 Bradyrhyzobium japonicum CCT 4065 + guar 0.0939

[0110] For the four strains, a higher value of bacteria growth rate is obtained in presence of guar. The addition of guar permits to increase the growth rate of these different strains of bacteria. In Table 2a are reported the relative increase of bacteria growth rate with the addition of guar compared to the control for each strain. An increase of bacteria growth rate between 2 and 45% is observed for the two gram positive bacteria (Bacillus subtilis and Bacillus megaterium), whereas a relative increase between 5 and 34% is observed for the two gram negative bacteria (Bradyrhyzobium japonicum and Agrobacterium radiobacter).

TABLE-US-00002 TABLE 2a Relative increase of bacteria growth rate with Strain guar addition Bacillus subtilis CCT 0089  2% Bacillus megaterium CCT 0536 45% Agrobacterium radiobacter CCT 4774 34% Bradyrhyzobium japonicum CCT 4065  5%

Example 1b

[0111] Another set of experiments was carried out, in which the ratio of microorganisms and guar was equal to 1.0×10.sup.10 CFU/g.

The bacteria growth rate (μ) obtained for the different experiments are summarized in Table 1b:

TABLE-US-00003 TABLE lb Bacteria growth Composition rate (h.sup.−1) Bacillus subtilis CCT 0089 0.0862 Bacillus subtilis CCT 0089 + guar 0.1172 Bacillus megaterium CCT 0536 0.0835 Bacillus megaterium CCT 0536 + guar 0.0912 Agrobacterium radiobacter CCT 4774 0.0882 Agrobacterium radiobacter CCT 4774 + guar 0.1102 Bradyrhyzobium japonicum CCT 4065 0.0915 Bradyrhyzobium japonicum CCT 4065 + guar 0.0897

[0112] For the four strains, a higher or comparable value of bacteria growth rate is obtained in presence of guar. For three of the bacteria strains, the addition of guar permits to increase the growth rate. In Table 2b are reported the relative increase of bacteria growth rate with the addition of guar compared to the control for each strain. An increase of bacteria growth rate between 9 and 36% is observed for the two gram positive bacteria (Bacillus subtilis and Bacillus megaterium), whereas this relative increase is equal to 25% for Agrobacterium radiobacter (gram negative bacteria). For Bradyrhyzobium Japonicum, a comparable growth rate is obtained with the addition of guar compared to control, hence the addition of guar maintains the growth rate of microorganisms.

TABLE-US-00004 TABLE 2b Relative increase of bacteria growth rate with Strain guar addition Bacillus subtilis CCT 0089 36% Bacillus megaterium CCT 0536  9% Bacillus megaterium CCT 4774 25% Bradyrhyzobium japonicum CCT 4065 ~0%

Example 2

[0113] The following materials are used in the experiments:
Guar: Cyamopsis tetragonoloba (guar) gum, available from Solvay (provided as a powder)
All microorganisms strains were acquired from Tropical Culture Collection in André Tosello Foundation—Brazil, some of them have reference in American Type Culture Colection (ATCC). [0114] Trichoderma harzianum CCT 4790 [0115] Aspergillus niger ATCC 16404 [0116] Beauveria bassiana ATCC 7159/DSM 1344
All strains were stored at −80° C. in the appropriate culture media, containing 20% of glycerol.
Culture media used in the experiments: [0117] Nutrient broth (NA) media containing per liter: 3 g of meat extract, 5 g of peptone and 15 g of agar (for solid media only) [0118] Oatmeal Agar (OA) containing per liter: 25 g of oat flakes or flour and 15 g of agar [0119] Malt Extract Agar 2% (MA2) containing per liter: 20 g of malt extract and 15 g of agar [0120] Sabouraud dextrose agar (SDA) containing per liter: 40 g of glucose, 10 g of peptone and 20 g agar
The media SDA, OA, and MA2 were used for reactivation of the strains A. niger, T. harzianum and B. bassiana, respectively, according to supplier's recommendation.
For the experiments with guar, only NA media was used.

Reactivation of Microorganisms:

[0121] A Petri dish containing 20 mL of SDA, OA or MA2 media was used for the reactivation of the strains A. niger, T. harzianum and B. bassiana, respectively. The stock culture was used to inoculate the solid media for each strain and the petri dishes were incubated at 25° C. until complete growth.
Incubation with Guar:
From the reactivation media on petri dish, the spores of fungi were recovered and a spore solution was prepared.
500 μL of the spore solution (approximately 1×10.sup.10 CFU/mL) were transferred to Erlenmeyer flasks containing 50 mL of media (controls and NA media with guar) and incubated at 25° C. Samples were taken at 48 h, 120 h and 168 h, filtered on filter paper and incubated at 60° C. before weighing [0122] Control media=NA without guar addition

Growth Evaluation:

[0123] The dry biomass recovery after each sample was plotted in a graphic dry biomass vs time and the growth curve could be obtained.
The growth rate (μ) was calculated considering only the exponential phase of the growth and compared with the control.
The μ value was used to compare all the experiments and to evaluate the influence of guar addition on fungi growth. The microorganisms growth rate (μ) obtained for the different experiments are summarized in Table 3:

TABLE-US-00005 TABLE 3 Fungi growth Composition rate (h.sup.−1) Trichoderma harzianum CCT 4790 0.0009 Bradyrhyzobium japonicum CCT 4790 + guar 0.0022 Aspergillus niger ATCC 16404 0.0008 Aspergillus niger ATCC 16404 + guar 0.0052 Beauveria bassiana ATCC 7159/DSM 1344 0.0870 Beauveria bassiana ATCC 7159/DSM 1344 + guar 0.1120
For the three strains, a higher growth rate is obtained in presence of guar. The addition of guar permits to increase the growth rate of these different strains of fungi. In Table 4 are reported the relative increase of fungi growth rate with the addition of guar compared to the control for each strain. An increase of fungi growth rate between 29 and 550% is observed for the three strains of fungi.

TABLE-US-00006 TABLE 4 Relative increase of fungi growth rate with Strain guar addition Trichoderma harzianum CCT 4790 144% Aspergillus niger ATCC 16404 550% Beauveria bassiana ATCC 7159/DSM 1344  29%

Example 3

[0124] The following materials are used in the experiments:
Guar: Cyamopsis tetragonoloba (guar) gum, available from Solvay (provided as a powder)
Bacteria strains were acquired from Tropical Culture Collection in André Tosello Foundation—Brazil. [0125] Bacillus thuringiensis CCT 2335 [0126] Pseudomonas putida CCT 5357
All strains were stored at −80° C. in the appropriate culture media, containing 15% of glycerol.
Only one culture media was used for both strains [0127] NA media containing per liter: 3 g of meat extract, 5 g of peptone and 15 g of agar (for solid media only)
A 250 mL shake flask containing 100 mL of NA culture media (reactivation media), was inoculated with 1 mL of the stock culture and incubated at 30° C., 150 rpm for 72 hours.
10 mL of this reactivation media were then transferred into a 250 mL shake flask containing 100 ml of culture media, with the addition of guar powder and incubated at 30° C., 150 rpm, for 96 hours.
An experiment without addition of guar powder is also performed for each strain as a control.
100 μL samples of each experiment were taken after 0 h, 24 h, 48 h, 72 h and 96 h of incubation.
These samples were diluted (the dilutions were variable according to strain growth, being from 1×10.sup.−5 up to 1×10.sup.−15) and the dilutions plated in solid NA media. The plates were incubated at 30° C. until appearance of colonies. After incubation, the number of colonies present in each dilution was counted and used to evaluate bacterial growth.
For bacterial growth rate determination, a graph of the log.sub.10(number of colonies) versus time of incubation was constructed. The straight line in this graph represents the exponential phase of bacterial growth and the angular coefficient represents the bacterial growth rate (μ).
The μ value was used to compare all the experiments and to evaluate the influence of guar addition on bacterial growth. For this set of experiments the ratio of microorganisms and guar is equal to 1.0×10.sup.5 CFU/g. The bacteria growth rate (μ) obtained for the different experiments are summarized in Table 5:

TABLE-US-00007 TABLE 5 Bacteria growth Composition rate (h.sup.−1) Bacillus thuringiensis CCT 2335 0.0898 Bacillus thuringiensis CCT 2335 + guar 0.1047 Pseudomonas putida CCT 5357 0.1133 Pseudomonas putida CCT 5357 + guar 0.1330
For the two strains, a higher value of bacteria growth rate is obtained in the presence of guar. Hence, the addition of guar permits to increase the bacteria growth rate. In Table 6 are reported the relative increase of bacteria growth rate with the addition of guar compared to the control for each strain. An increase of bacteria growth rate equals to 17% is observed for the two strains of bacteria.

TABLE-US-00008 TABLE 6 Relative increase of bacteria growth rate with Strain guar addition Bacillus thuringiensis CCT 2335 17% Pseudomonas putida CCT 5357 17%