METHOD FOR PROLONGING THE VIABILITY OF FUNGAL SPORES IN LIQUID FORMULATIONS

Abstract

The present invention relates to a method for prolonging spore viability of fungal spores present in a liquid formulation, comprising (a) packaging said formulation in a suitable container; (b) reducing oxygen exposure of the spores in said formulation as compared to oxygen exposure of said spores when said formulation is in contact with air; and (c) closing or sealing said container. The invention further relates to a closed container comprising a liquid formulation within said container, said formulation comprising fungal spores, wherein the spores in said formulation have reduced oxygen exposure as compared to oxygen exposure of said spores when said formulation is in contact with air.

Claims

1. A method for prolonging spore viability of fungal spores present in a liquid formulation, comprising (a) packaging said formulation in a suitable container; (b) reducing oxygen exposure of the spores in said formulation as compared to oxygen exposure of said spores when said formulation is in contact with air; and (c) closing or sealing said container.

2. The method of claim 1, wherein said container is with reduced or without head space.

3. The method of claim 1, wherein said container reduces or prevents diffusion of oxygen.

4. The method of claim 1, wherein said reducing oxygen exposure in step (b) is effected by reducing oxygen in the head space of said container.

5. The method of claim 1, wherein reducing oxygen exposure in step (b) is effected by evacuating said container.

6. The method of claim 1, wherein reducing oxygen exposure in step (b) is effected by evacuating said container and subsequent refilling with an inert gas or mixtures of inert gases.

7. The method of claim 1, reducing oxygen exposure in step (b) is effected by (i) providing an oxygen-absorbing agent at the inside of the container, (ii) introducing an oxygen-absorbing agent into the container and subsequently sealing said container or (iii) including an oxygen-absorbing agent into the container material; and subsequently sealing said container after filling it with the formulation.

8. The method of claim 6, wherein step (b) is repeated at least twice.

9. The method of claim 4, wherein said oxygen exposure in step (b) is reduced by flushing said head space with an inert gas or a mixture of gases comprising less oxygen compared to air or no oxygen.

10. The method of claim 6, wherein said gas is hydrogen, nitrogen, helium, neon, argon, krypton, xenon, radon, carbon dioxide, nitrous oxide, hydrogen sulfide, lower alkane or halo alkane or mixtures thereof.

11. The method of claim 6, wherein said gas is nitrogen.

12. The method of claim 1, wherein said formulation is essentially free of water.

13. The method of claim 1, wherein said spores are from a fungal species selected from Purpureocillium lilacinum, Isaria fumosorosea, Beauveria bassiana, Cladosporium cladosporioides, Clonostachys rosea, Coniothyrium minitans, Metarhizium anisopliae, Nomurea rileyi, Penicillium bilaii, Simplicillium lanosoniveum and Talaromyces flavus.

14. A closed container comprising a liquid formulation within said container, said formulation comprising fungal spores, wherein the spores in said formulation have reduced oxygen exposure as compared to oxygen exposure of said spores when said formulation is in contact with air.

15. The container of claim 14, wherein said spores are from a fungal species selected from Purpureocillium lilacinum, Isaria fumosorosea, Beauveria bassiana, Cladosporium cladosporioides, Clonostachys rosea, Coniothyrium minitans, Metarhizium anisopliae, Nomurea rileyi, Penicillium bilaii, Simplicillium lanosoniveum and Talaromyces flavus.

16. The container of claim 15, wherein said spores are from Purpureocillium lilacinum.

17. The container of claim 16, wherein said Purpureocillium lilacinum is strain 251.

18. The container of claim 14, wherein said formulation is essentially free of water.

19. A method of combatting plant pests or phytopathogenic fungi comprising providing the container according to claim 14, preparing the formulation contained therein for agricultural use or for use as a biocide and applying said prepared formulation to a plant or a spot in need thereof.

20. Use of the formulation comprised in the container of claim 14 in agriculture or as a biocide.

Description

EXAMPLE 1

Comparison of Packaging Materials Allowing for Oxygen Diffusion or Being Airtight

[0090] 100 mL of the formulation comprising a polyether-modified trisiloxane, fumed silica and spores of Purpureocillium lilacinum were filled in bottles (HDPE or Coex(PE/EVOH) respectively, nominal filling volume 100 mL) and sealed. The sealed bottles were then stored in temperature controlled storage cabinets (4° C., 30° C. and 40° C.) for a defined time. Before determination of viability, the bottles were equilibrated to ambient temperature. The viability was determined using a microbiological counting method. The values listed in the following tables are illustrated in FIGS. 1a to 1c.

EXAMPLE 1a:

[0091]

TABLE-US-00001 Storage temperature: 4° C. Sample taken at/after HDPE Coex (PE/EVOH) day 0 94.4% 94.4% 8 weeks 87.3% 88.5%

EXAMPLE 1b:

[0092]

TABLE-US-00002 Storage temperature: 30° C. Sample taken at/after HDPE Coex (PE/EVOH) day 0 94.4% 94.4% 8 weeks 55.2% 68.1%

EXAMPLE 1c:

[0093]

TABLE-US-00003 Storage temperature: 40° C. Sample taken at/after HDPE Coex (PE/EVOH) day 0 94.4% 94.4% 8 weeks 0.0% 19.1%

EXAMPLE 2

Comparison of Sealed Bottles With Atmospheric and Reduced Oxygen Content

[0094] 100 mL of the formulation comprising a polyether-modified trisiloxane, fumed silica and spores of Purpureocillium lilacinum were filled in coextruded bottles (PE/EVOH, nominal filling volume 100 mL) and sealed with or without prior nitrogen flushing. In case of directly sealing the bottle, the initial oxygen content in the headspace of the bottle reflects the atmospheric oxygen content of ca. 21%. In the case of flushing the filled bottle with nitrogen prior to sealing, the oxygen content was reduced by at least 50%. This was established by pre-trial where filled bottles where flushed with nitrogen and sealed and the oxygen content of the sealed bottle immediately determined by use of an oxygen sensor. The sealed bottles were then stored in temperature controlled storage cabinets (20° C., 25° C., 30° C. and 35° C.) for a defined time. Before determination of viability, the bottles were equilibrated to ambient temperature. The viability was determined using a microbiological counting method. The values listed in the following tables are illustrated in FIGS. 2a to 2d.

EXAMPLE 2a:

[0095]

TABLE-US-00004 Storage temperature: 20° C. Sample taken at/after N2 flushing: no N2 flushing: yes day 0 98% 98% 3 month 95% 97% 6 month 80% 93%

EXAMPLE 2b:

[0096]

TABLE-US-00005 Storage temperature: 25° C. Sample taken at/after N2 flushing: no N2 flushing: yes day 0 98% 98% 3 month 90% 90% 6 month 62% 85%

EXAMPLE 2c:

[0097]

TABLE-US-00006 Storage temperature: 30° C. Sample taken at/after N2 flushing: no N2 flushing: yes day 0 98% 98% 3 month 69% 89% 6 month 19% 71%

EXAMPLE 2d:

[0098]

TABLE-US-00007 Storage temperature: 35° C. Sample taken at/after N2 flushing: no N2 flushing: yes day 0 98% 98% 3 month 39% 77%

EXAMPLE 3

Comparison of Packaging Materials Under Reduced Oxygen Content

[0099] Under an atmosphere containing ca. 8% oxygen in nitrogen, approx. 40 mL of the formulation described in Examples 1 and 2 were filled in co-extruded bottles (HDPE/EVOH or HDPE/PA respectively, nominal filling volume 50 mL) and sealed. The sealed bottles were then stored in temperature controlled storage cabinets for a defined time. Before determination of viability, the bottles were equilibrated to ambient temperature. The viability was determined using a microbiological counting method. The values listed in the following tables are illustrated in FIGS. 3a to 3b.

EXAMPLE 3a

[0100]

TABLE-US-00008 Storage temperature: 4° C. Sample taken at/after Coex (PE/EVOH) Coex (PE/PA) day 0 97.7% 97.7% 3 months 97.1% 97.4% 6 months 96.3% 96.5%

EXAMPLE 3b

[0101]

TABLE-US-00009 Storage temperature: 20° C. Sample taken at/after Coex (PE/EVOH) Coex (PE/PA) day 0 97.7% 97.7% 3 months 96.3% 96.8% 6 months 95.3% 94.0%

[0102] As can be seen from the tables above, both kinds of co-extruded materials can be used in the present invention.

EXAMPLE 4

Comparison of Head Space Volume

[0103] Co-extruded bottles (HDPE/EVOH, nominal filling volume 50 mL) were filled with the formulation under air atmosphere (oxygen content of ca. 21%) to 100%, 90% and 80% respectively of the total bottle volume and sealed. The sealed bottles were then stored in temperature controlled storage cabinets for a defined time. Before determination of viability, the bottles were equilibrated to ambient temperature. The viability was determined using a microbiological counting method. The values listed in the following tables are illustrated in FIG. 4.

TABLE-US-00010 Storage temperature: 20° C. Sample taken at/after 0% headspace 10% headspace 20% headspace day 0 96.9% 96.9% 96.9% 3 months 95.9% 96.5% 96.7% 6 months 94.8% 93.2% 91.8%

[0104] This examples shows that reducing head space is one means of realizing the present invention.

EXAMPLE 5

Comparison of Reduced Oxygen Contents in Headspace

[0105] Co-extruded bottles (HDPE/EVOH, nominal filling volume 50 mL) were filled with approx. 40 mL of the formulation under atmospheres containing different concentrations of oxygen in nitrogen (0%, 2%, 4%, 6%, 8% oxygen at time of filling) and sealed. The sealed bottles were then stored in a temperature controlled storage cabinet for a defined time. Before determination of viability, the bottles were equilibrated to ambient temperature. The viability was determined using a microbiological counting method. The values listed in the following tables are illustrated in FIG. 5.

TABLE-US-00011 Storage temperature: 35° C. Sample taken 0% 2% 4% 6% 8% at/after oxygen oxygen oxygen oxygen oxygen day 0 97.0% 96.7% 96.1% 96.2% 97.7% 1 months 94.3% 92.9% 94.4% 95.4% 92.6% 3 months 95.6% 95.1% 94.6% 93.2% 90.7% 6 months 92.9% 93.5% 89.6% 81.2% 72.0%

[0106] As can be seen from the table above, reducing the oxygen content in the headspace prolongs shel-life of the fungal spores.

EXAMPLE 6

Comparison of Inert Gases

[0107] Co-extruded bottles (HDPE/EVOH, nominal filling volume 50 mL) were filled with approx. 40 mL of the formulation under an atmosphere of argon and nitrogen respectively containing ca. 0% oxygen at time of filling and sealed. The sealed bottles were then stored in temperature controlled storage cabinets for a defined time. Before determination of viability, the bottles were equilibrated to ambient temperature. The viability was determined using a biochemical counting method. The values listed in the following tables are illustrated in FIG. 6.

TABLE-US-00012 Storage temperature: 20° C. Sample taken at/after argon nitrogen day 0 97.0% 97.0% 1 month 96.7% 96.8% 3 months 96.4% 97.0% 6 months 95.8% 94.9%

EXAMPLE 7

Comparison of Inert Gases Mixtures

[0108] Co-extruded bottles (HDPE/EVOH, nominal filling volume 50 mL) were filled with approx. 40 mL of the formulation under an atmosphere of carbon dioxide and oxygen in nitrogen and sealed. After one day the content of carbon dioxide and oxygen in the headspace of one replicate of the sealed bottles was determined. The other replicates of the sealed bottles were then stored in temperature controlled storage cabinets for a defined time. Before determination of viability, the bottles were equilibrated to ambient temperature. The viability was determined using a microbiological counting method. The values listed in the following tables are illustrated in FIG. 7.

TABLE-US-00013 Storage temperature: 20° C. Sample taken ca. 8% oxygen/22% ca. 9% oxygen/5% at/after carbon dioxide in nitrogen carbon dioxide in nitrogen day 0 .sup. 97%   97% 1 month 97.1% Not determined 3 months 97.0% 96.8% 6 months 94.6% 95.2%

[0109] The table above shows that also mixtures of inert gases provide for the effect of prolonging shelf-life of fungal spores in liquid formulations.