NOVEL CARRIER FLUIDS FOR LIQUID FUNGAL SPORE FORMULATIONS

Abstract

The present invention relates to a liquid preparation comprising at least one carboxylic ester as defined and fungal spores and a method for controlling phytopathogenic fungi, insects and/or nematodes in or on a plant, for enhancing growth of a plant or for increasing plant yield or root health comprising applying an effective amount of the liquid preparation or the liquid composition according to the invention to said plant or to a locus where plants are growing or intended to be grown.

Claims

1. A liquid preparation comprising at least one carboxylic ester composed of a carboxylic acid moiety and an alcohol moiety wherein said carboxylic ester is not a carboxylic acid triglyceride from vegetable oils, and fungal spores of a fungus exerting a beneficial effect on plants, wherein said at least one carboxylic ester contains a) a carboxylic monoacid moiety and a monoalcohol moiety b) at least one carboxylic monoacid moiety and a polyalcohol moiety or c) a carboxylic polyacid moiety and at least one monoalcohol moiety; wherein said monoalcohol moiety is a branched, linear, cyclic, acyclic or partially cyclic, saturated or partially unsaturated C1-C24 monoalcohol moiety; wherein said carboxylic monoacid moiety is a branched, linear, cyclic, acyclic or partially cyclic, saturated or partially unsaturated C2-C24 carboxylic monoacid moiety, optionally carrying at least one OH functionality; wherein said polyalcohol moiety is a branched, linear, cyclic, acyclic or partially cyclic, saturated or partially unsaturated di-, tri-, tetra-, penta- or hexavalent C2-C20 polyalcohol moiety; and wherein said carboxylic polyacid moiety is a branched, linear, cyclic, acyclic or partially cyclic, saturated or partially unsaturated C2-C20 carboxylic polyacid moiety.

2. The liquid preparation according to claim 1, wherein any one of a), b) and/or c) is a mixture of esters comprised of more than one different monoalcohol moiety, polyalcohol moiety, carboxylic monacid moiety or carboxylic polyacid moiety.

3. The liquid preparation according to claim 1, comprising a mixture of carboxylic esters according to any one of a) to c).

4. The liquid preparation according to claim 1, wherein said monoalcohol moiety is derived from a branched, linear, saturated or partially unsaturated C1-C20 monoalcohol selected from the group consisting of methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, 1-pentanol, 1-hexanol, 1-heptanol, 2-ethylhexan-1-ol, capryl alcohol, pelargonic alcohol, isononyl alcohol, capric alcohol, undecanol, lauryl alcohol, tridecanol, isotridecanol, myristyl alcohol, pentadecanol, cetyl alcohol, palmitoleyl alcohol, heptadecanol, stearyl alcohol, oleyl alcohol, nonadecanol, eicosanol, and mixtures thereof.

5-6. (canceled)

7. The liquid preparation according to claim 1, wherein said at least one carboxylic monoacid moiety is derived from a branched, linear, saturated or partially unsaturated C2-C20 carboxylic acid selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, isostearic acid, oleic acid, linoleic acid, α-linolenic acid, ricinolic acid and mixtures thereof.

8. (canceled)

9. The liquid preparation according to claim 1, wherein said at least one polyalcohol moiety is derived from a polyalcohol selected from the group consisting of glycol, 1,3-propandiol, 1-4-butandiol, 1,5-pentandiol, 1,6-hexandiol, cyclohexan-1,2-diol, isosorbid, 1,2-propandiol, neopentylglycol, glycerol, trimethylolpropane, pentaerythritol and sugar alcohols.

10. (canceled)

11. The liquid preparation according to claim 1, wherein said at least one carboxylic polyacid moiety is derived from a carboxylic polyacid selected from the group consisting of (a) a linear, saturated or partially unsaturated C2-C10 dicarboxylic acid (b) a cyclic C5-C6 dicarboxylic acid, and (c) citric acid and its O-acylated derivatives, such as 0-acetyl citric acid.

12-13. (canceled)

14. The liquid preparation according to claim 1, wherein said at least one carboxylic monoacid moiety or at least one carboxylic polyacid moiety carries at least one OH functionality.

15. The liquid preparation according to claim 1, wherein said at least one polyalcohol moiety of said at least one carboxylic ester according to b) is partially or fully esterified.

16. (canceled)

17. The liquid preparation according to claim 1, wherein said at least one carboxylic ester according to a) comprises between 13 and 28 carbon atoms.

18. The liquid preparation according to claim 1, wherein said carboxylic monoacid moiety is derived from a carboxylic monoacid selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, isostearic acid, oleic acid, linoleic acid, α-linolenic acid, ricinolic acid and mixtures thereof.

19. The liquid preparation according to claim 18, wherein said monoalcohol moiety is derived from a monoalcohol selected from the group consisting of methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, 1-pentanol, 1-hexanol, 1-heptanol, 2-ethylhexan-1-ol, capryl alcohol, pelargonic alcohol, isononyl alcohol, capric alcohol, lauryl alcohol, tridecanol, isotridecanol, myristyl alcohol, cetyl alcohol, palmitoleyl alcohol, stearyl alcohol, oleyl alcohol and mixtures thereof.

20-21. (canceled)

22. The liquid preparation according to claim 1, wherein said carboxylic ester is selected from the group consisting of 2-ethylhexyl laurate, 2-ethylhexyl palmitate, 2-ethylhexyl oleate, ricinolic acid methylester and propionic acid pentyl ester.

23. The liquid preparation according to claim 1, wherein in said at least one carboxylic ester according to b), said carboxylic monoacid moiety is derived from a carboxylic monoacid selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, isostearic acid, oleic acid, linoleic acid, α-linolenic acid and ricinolic acid and said polyalcohol moiety is derived from a polyalcohol selected from the group consisting of 1,2-ethandiol, 1,3-propandiol, 1-4-butandiol, 1,5-pentandiol, 1,6-hexandiol, cyclohexan-1,2-diol, isosorbid, 1,2-propandiol, neopentylglycol, glycerol, pentaerythritol, trimethylolpropan and sugar alcohols.

24. The liquid preparation according to claim 1, wherein in said at least one carboxylic ester according to b), said carboxylic monoacid moiety is a branched, linear, cyclic, acyclic or partially cyclic, saturated or partially unsaturated C2-C6 carboxylic monoacid moiety, optionally carrying at least one OH functionality, preferably a C2 to C5 carboxylic monoacid moiety.

25. The liquid preparation according to claim 1, wherein in said at least one carboxylic ester according to b), said polyalcohol moiety is a cyclic or partially cyclic, saturated or partially unsaturated C2-C20-divalent, C3-C20-trivalent, C4-C20-tetravalent, C-5-C20-pentavalent or C6-C20-hexavalent polyalcohol moiety; or a polyalcohol of the following formula II ##STR00003## where n is an integer between 0 and 4, where R1 and R2 are independent from each other hydrogen or hydroxy, where R2 is C1-C9 alkyl if n=1 and R1=OH wherein said cyclic or partially cyclic, saturated or partially unsaturated C2-C20-divalent, C3-C20-trivalent, C4-C20-tetravalent, C-5-C20-pentavalent or C6-C20-hexavalent polyalcohol moiety is derived from a sugar alcohol and wherein said sugar alcohol is selected from the group consisting of ethylene glycol, propylene glycol, glycerol, erythrol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriol, maltotetraitol, polyglycitol and sorbitan.

26-28. (canceled)

29. The liquid preparation according to claim 23, wherein said polyalcohol is glycerol and said carboxylic monoacid is selected from the group consisting of acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid and/or capric acid or mixtures thereof.

30. The liquid preparation according to claim 23, wherein said carboxylic ester is composed of diacetylglycerol esterified with acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, isostearic acid, oleic acid, linoleic acid, α-linolenic acid and ricinolic acid.

31. The liquid preparation according to claim 1, wherein said carboxylic ester is selected from the group consisting of proplylene glycol dicaprylate, propylene glycol dicaprate, neopentylglycol dicocoate, glycerol triacetate, trimethylolpropane triisostearate, trimethylolpropane tricocoate, glycerol tricaprylate, glycerol tricaprate, C12-C18 carboxylic acid monoglyceride diacetate, trimethylolpropane tricaprylate, trimethylolpropane tricaprate, trimethylolpropane trioleate and sorbitan trioleate.

32. The liquid preparation according to claim 1, wherein in said at least one carboxylic ester according to c), said carboxylic polyacid moiety is derived from linear, saturated or partially unsaturated C2-C10 dicarboxylic acids, cyclic C5-C6 dicarboxylic acids, citric acid and its 0-acetylated derivatives and wherein said monoalcohol moiety is derived from a monoalcohol selected from the group consisting of methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, pentan-1-ol, pentan-2-ol, pentan-3-ol, 2-methylbutan-1-ol, 2-methylbutan-2-ol, 3-methylbutan-1-ol, 3-methylbutan-2-ol, 2,2-dimethylpropan-1-ol, 1-hexanol, 1-heptanol, 2-ethylhexan-1-ol, capryl alcohol, pelargonic alcohol, isononyl alcohol, capric alcohol, lauryl alcohol, tridecanol, isotridecanol, myristyl alcohol, cetyl alcohol, palmitoleyl alcohol, stearyl alcohol and oleyl alcohol.

33-36. (canceled)

37. The liquid preparation according to claim 1, wherein said fungal spores are from a fungal species which is effective as biological control agent in plant protection or plant health promoting agent.

38. The liquid preparation according to claim 37, wherein said fungal species is an entomopathogenic fungus selected from the group consisting of Isaria fumosorosea, Penicillium bilaii, Metarhizium anisopliae, Purpureocillium lilacinum, Coniothyrium minitans, Beauveria bassiana and Clonostachys rosea.

39-40. (canceled)

41. The liquid preparation according to claim 1, further comprising at least one substance selected from the group consisting of surfactants, rheology modifiers, antifoaming agents, antioxidants and dyes.

42. A liquid composition comprising the liquid preparation according to claim 1.

43. A method for controlling phytopathogenic fungi, insects and/or nematodes in or on a plant, for enhancing growth of a plant or for increasing plant yield or root health comprising applying an effective amount of the liquid preparation of claim 1 to said plant or to a locus where plants are growing or intended to be grown.

44. (canceled)

Description

EXAMPLE I (PURPUREOCILLIUM LILACINUM)

[0131] 3 g of Purpureocillium lilacinum pure spore powder were transferred into a formulation vessel (IKA Type DT-20 mixing vessel with dispersion tool for Ultra Turrax) using a sterile spoon. 12 mL of fluid were added into the respective formulation vessel and dispersed using ultra turrax tube drive control for 1 min at 3000 rpm; change direction after 30 sec. After this 2.8 mL were transferred in four sample bottles (Wheaton Serum vial, Type I) leaving little headspace and closed tight using crimpneck caps (Macherey—Nagel type N 13). Afterwards all sample bottles were transferred to an incubator set at 30° C. and stored for a given time.

[0132] In regular intervals a sample was retrieved from the storage location and analyzed for spore viability. For this purpose, the original samples were thoroughly homogenized. Aliquots of 0.25 g or 25 μL of each sample were transferred into 50 mL falcon tubes. The tubes were filled up to 25 g using a sterile aqueous solution containing 2% Tween 80 and homogenized by vortexing to achieve the first dilution step (1:100 dilution). This dilution was used for further dilution and spotting on agar.

[0133] Not all samples mixed well or mixed at all in 2% Tween 80. For these samples, an alternative dispersion/dilution method was applied where the oil phase was stripped from the spores first: 0.25 g or 250 μL of sample are weighted into a 2 mL Eppendorf tube, and 0.5 mL of 2% Tween 80 is added, and the mixture is transferred into an Eppendorf centrifuge where it is centrifuged for 1 min at 10.000 rpm. The supernatant (=upper oily phase) is discarded by using a pipette. Afterwards 250 μL of Breakthru S 240 were added and the spores were well dispersed. 250 μL or 0.25 g of each sample were transferred into a sterile 50 mL Falcon tube. The tubes were filled up to 25 g using a sterile aqueous solution containing 2% Tween 80 and homogenized by vortexing to achieve the first dilution step (1:100 dilution). This dilution was used for further dilution and spotting on agar.

[0134] For evaluation of spore germination rate prepare a 1:30000 dilution based on the 1:100 dilution achieved by multiple automated dilution (pipetting robot, 96 well plate). Afterwards 12×12 cm agar plates were taken and spotted with 10 times 54 of each sample using an automated 12-Channel pipet. Wait until liquid is soaked up by agar and transfer agar plate to an incubator and incubate at 25° C. for 17 hours. Open the plate and place it under the microscope. Randomly chose one area per spot and record the number of germinated and non-germinated spores that are within the designated field. At least 200 spores per sample need to be evaluated. If needed count more than one field per spot.

[0135] The results of spore viabilities are given in table I.

TABLE-US-00003 TABLE I spore viability [%] Liquid at day after 2 w after 1 m after 5-6 w after 2 m after 3 m # Liquid Class 1 (12-15 d) (27-28 d) (35-40 d) (51-55 d) (87-92 d) 1 Pentyl Propionate a 99.1 94.6 16.1 2 Radia 7081 a n/a.sup.# 94.7 95.8 88.6 3 Radia 7127 a 99.1 99.0 98.3 88.4 40.6* 4 Radia 7331 a n/a.sup.# 97.9 95.9 85.9 5 Radialube 7130 a n/a.sup.# 98.5 94.3 87.9 6 Radia 7208 b n/a.sup.# 95.7 98.3 92.9 7 Radia 7368 b 98.6 96.9 89.6 8 Radia 7380 b 96.5 89.3 86.0 9 Radia 7909 b 98.1 96.5 78.5 10 Radialube 7302 b 99.1 94.7 83.1 11 Radialube 7359 b .sup. 58.0.sup.# 60.6.sup.# 87.8 87.5 87.3* 12 Radialube 7361 b n/a.sup.# 97.2 92.3 85.4 13 Radiasurf 7355 b 98.3 96.4 64.1 14 Triacetin b 98.3 97.9 96.2 90.8 69.4* 15 Acetyltributylcitrat c 98.9 98.0 87.5 16 Adimoll DB c 98.9 97.5 89.8 17 Agnique AE829 c n/a.sup.# 97.3 96.5 89.8 18 Crodamol DA c 98.2 94.3 95.0 68.2 51.3* 19 Breakthru S240.sup.$ 90.5 91.8 85.8 76.8 7.4* 20 Mero EC ®.sup.$ 93.9 89.4 4.5 *= taken after 7 m of storage; .sup.#= difficulties to disperse in water for evaluation; .sup.$= comparative examples

[0136] Discussion: Spore viability directly after manufacturing of the samples (day 1) is generally at or above 90% for the vast majority of all fluids tested. Fluids according to the invention exhibit a spore viability of approx. 70% or greater after storage for 2 or 3 months at 30° C., respectively. Selected fluids have been stored for 7 months at 30° C. and exhibit a spore viability of approx. 40% or greater after storage (Table I, entries 3,11,14,18,19). BreakThru 5240 has been previously described as a superior fluid to host fungal spores. For comparison, BreakThru 5240 (Table I, entry 19) provides ˜77% spore viability after 2m of storage and approx. 7% after 7m of storage under the given test conditions. Mero EC® (Table I, entry 20), which is a tankmix additive that serves as a comparative example for self-emulsifying methylated seed oils, exhibited only a marginal spore viability of approx. 5% after 3m of storage.

EXAMPLE II (ISARIA FUMOSOROSEA)

[0137] 1.5 g of I. fumosorosea pure spore powder were transferred into a formulation vessel (IKA Type DT-20 mixing vessel with dispersion tool for Ultra Turrax) using a sterile spoon. 13.5 mL of fluid were added into the respective formulation vessel and dispersed using ultra turrax tube drive control for 1 min at 3000 rpm; change direction after 30 sec. After this 2.8 mL were transferred in four sample bottles (Wheaton Serum vial, Type I) leaving little headspace and closed tight using crimpneck caps (Macherey—Nagel type N 13) Afterwards all sample bottles were transferred to an incubator set at 30° C. and stored for a given time.

[0138] In regular intervals a sample was retrieved from the storage location and analyzed for spore viability. Therefore the original samples were thoroughly homogenized. Aliquots of 0.25 g or 25 μL of each sample were transferred into 50 mL falcon tubes. The tubes were filled up to 25 g using a sterile aqueous solution containing 2% Tween 80 and homogenized by vortexing to achieve the first dilution step (1:100 dilution). This dilution was used for further dilution and spotting on agar.

[0139] Not all samples mixed well or mixed at all in 2% Tween 80. For these samples, an alternative dispersion/dilution method was applied where the oil phase is stripped from the spores first: 0.25 g or 250 μL of sample were weighted into a 2 mL Eppendorf tube, and 0.5 mL of 2% Tween 80 was added, and the mixture was transferred into an Eppendorf centrifuge where it was centrifuged for 1 min at 10.000 rpm. The supernatant (=upper oily phase) was discarded by using a pipette. Afterwards 250 of Breakthru S 240 were added and the spores were well dispersed. 250 μL or 0.25 g of each sample were transferred into a sterile 50 mL Falcon tube. The tubes were filled up to 25 g using a sterile aqueous solution containing 2% Tween 80 and homogenized by vortexing to achieve the first dilution step (1:100 dilution). This dilution was used for further dilution and spotting on agar.

[0140] For evaluation of spore germination rate prepare a 1:15000 dilution based on the 1:100 dilution achieved by multiple automated dilution (pipetting robot, 96 well plate). Afterwards 12×12 cm agar plates are taken and spotted with 10 times 54 of each sample using an automated 12-Channel pipet. Wait until liquid is soaked up by agar and transfer agar plate to an incubator and incubate at 23° C. for 16 hours. Open the plate and place it under the microscope. Randomly chose one area per spot and record the number of germinated and non-germinated spores that are within the designated field. At least 200 spores per sample need to be evaluated. If needed count more than one field per spot. The results of spore viabilities are given in table II.

TABLE-US-00004 TABLE II spore viability [%] after 1 m after 7 m # Liquid at day 1 (27 d) (210 d) 1 Catenex T 121.sup.$ 91.8 60.5 64.2 2 Adimoll DB 89.5 66.6 60.0 3 Triacetin 90 84.0 69.1 4 Breakthru S240.sup.$ 90.9 78.7 69.6 .sup.$= comparative examples

Discussion:

[0141] Spore viability directly after manufacturing of the spores (day 1) is generally around 90% for all fluids tested. Fluids according to the invention exhibit a spore viability of approx. 60% or greater after storage for 7m at 30° C. (Table II, entries 2,3) and thus equal the performance level of the comparison examples, i.e. BreakThru 5240 and Catenex T 121 (Table 2 entries 1, 4).

EXAMPLE III (BEAUVERIA BASSIANA)

[0142] 1.5 g of Beauveria bassiana pure spore powder were transferred into a formulation vessel (IKA Type DT-20 mixing vessel with dispersion tool for Ultra Turrax) using a sterile spoon. 13.5 mL of fluid were added into the respective formulation vessel and dispersed using ultra turrax tube drive control for 1 min at 3000 rpm; change direction after 30 sec. After this 2.8 mL were transferred in four sample bottles (Wheaton Serum vial, Type I) leaving little headspace and closed tight using crimpneck caps (Macherey—Nagel type N 13) Afterwards all sample bottles were transferred to an incubator set at 30° C. and stored for a given time.

[0143] In regular intervals a sample was retrieved from the storage location and analyzed for spore viability. Therefore the original samples were thoroughly homogenized. Aliquots of 0.25 g or 25 μL of each sample are transferred into 50 mL falcon tubes. The tubes were filled up to 25 g using a sterile aqueous solution containing 2% Tween 80 and homogenized by vortexing to achieve the first dilution step (1:100 dilution). This dilution was used for further dilution and spotting on agar.

[0144] Not all samples mixed well or mixed at all in 2% Tween 80. For these samples, an alternative dispersion/dilution method was applied where the oil phase is stripped from the spores first: 0.25 g or 250 μL of sample are weighted into a 2 mL Eppendorf tube, and 0.5 mL of 2% Tween 80 is added, and the mixture is transferred into an Eppendorf centrifuge where it is centrifuged for 1 min at 10.000 rpm. The supernatant (=upper oily phase) is discarded by using a pipette. Afterwards 250 μL of Breakthru S 240 are added and the spores are well dispersed. 250 μL or 0.25 g of each sample are transferred into a sterile 50 mL Falcon tube. The tubes were filled up to 25 g using a sterile aqueous solution containing 2% Tween 80 and homogenized by vortexing to achieve the first dilution step (1:100 dilution). This dilution is used for further dilution and spotting on agar.

[0145] For evaluation of spore germination rate prepare a 1:15000 dilution based on the 1:100 dilution achieved by multiple automated dilution (pipetting robot, 96 well plate). Afterwards 12×12 cm agar plates are taken and spotted with 10 times 5 μL of each sample using an automated 12-Channel pipet. Wait until liquid is soaked up by agar and transfer agar plate to an incubator and incubate at 20° C. for 17 hours. Open the plate and place it under the microscope. Randomly chose one area per spot and record the number of germinated and non-germinated spores that are within the designated field. At least 200 spores per sample need to be evaluated. If needed count more than one field per spot. The results of spore viabilities are given in Table III.

TABLE-US-00005 TABLE III spore viability [%] at day after 2 w after 5 w # Liquid 1 (14 d) (35 d) 1 Radia 7127 91.3 76.3 68.4 2 Radia 7331 91.5 81.4 66.3 3 Adimoll DB 87 66.1 37.7 4 Agnique AE829 89.7 74.9 57.8 5 Radialube 7302 84.2 68.4 52.7 6 Radiasurf 7355 n/a.sup.# 71.3 60.4 7 Radia 7208 86.9 66.8 52.7 8 Radia 7368 90.6 80.8* 9 Triacetin 89.3 58.6 31.6 10 Radia 7909 75.1 11.5 18.1 11 Acetyltributylcitrat 85.5 76.7 58.8 12 Miglyol 812 90.9 74.1 49.0 13 Breakthru S240.sup.$ 53.1 27.6 19.6 *= taken after 21 d of storage; .sup.#= difficulties to disperse in water for evaluation; .sup.$= comparative example

[0146] Discussion: Spore viability directly after manufacturing of the samples (day 1) is generally high and in most cases at or above 75%, in many cases even close to or above 90% under the given test conditions Among the examples according to the invention are selected fluids that exhibit a spore viability of approx. 30% or greater after storage for 5w or longer at 30° C. Many fluids even provide a spore viability of ˜50% or greater, in some cases even ˜60% or greater (Table III, entries 1, 2, 4, 6, 11) For comparison, BreakThru 5240 provides only approx. 53% spore viability at day 1 and approx. 20% after 5w of storage (Table III, entry 13).

EXAMPLE IV: PENICILLIUM BILAII

[0147] 1.5 g of Penicillium bilaii pure spore powder were transferred into a formulation vessel (IKA Type DT-20 mixing vessel with dispersion tool for Ultra Turrax) using a sterile spoon. 13.5 mL of fluid were added into the respective formulation vessel and dispersed using ultra turrax tube drive control for 1 min at 3000 rpm; change direction after 30 sec. After this 2.8 mL were transferred in four sample bottles (Wheaton Serum vial, Type I) leaving little headspace and closed tight using crimpneck caps (Macherey—Nagel type N 13) Afterwards all sample bottles were transferred to an incubator set at 30° C. and stored for a given time.

[0148] In regular intervals a sample was retrieved from the storage location and analyzed for spore viability. Therefore the original samples were thoroughly homogenized. Aliquots of 0.25 g or 25 μL of each sample are transferred into 50 mL falcon tubes. The tubes were filled up to 25 g using a sterile aqueous solution containing 2% Tween 80 and homogenized by vortexing to achieve the first dilution step (1:100 dilution). This dilution was used for further dilution and spotting on agar.

[0149] Not all samples mixed well or mixed at all in 2% Tween 80. For these samples, an alternative dispersion/dilution method was applied where the oil phase is stripped from the spores first: 0.25 g or 250 μL of sample are weighted into a 2 mL Eppendorf tube, and 0.5 mL of 2% Tween 80 is added, and the mixture is transferred into an Eppendorf centrifuge where it is centrifuged for 1 min at 10.000 rpm. The supernatant (=upper oily phase) is discarded by using a pipette. Afterwards 250 μL of Breakthru S 240 are added and the spores are well dispersed. 250 μL or 0.25 g of each sample are transferred into a sterile 50 mL Falcon tube. The tubes were filled up to 25 g using a sterile aqueous solution containing 2% Tween 80 and homogenized by vortexing to achieve the first dilution step (1:100 dilution). This dilution is used for further dilution and spotting on agar.

[0150] For evaluation of spore germination rate prepare a 1:15000 dilution based on the 1:100 dilution achieved by multiple automated dilution (pipetting robot, 96 well plate). Afterwards 12×12 cm agar plates are taken and spotted with 10 times 54 of each sample using an automated 12-Channel pipet. Wait until liquid is soaked up by agar and transfer agar plate to an incubator and incubate at 20° C. for 17 hours. Open the plate and place it under the microscope. Randomly chose one area per spot and record the number of germinated and non-germinated spores that are within the designated field. At least 200 spores per sample need to be evaluated. If needed count more than one field per spot. The results of spore viabilities are given in Table IV.

TABLE-US-00006 TABLE IV spore viability [%] initial 6 w@30° C. 3 m@30° C. No Liquid (1-2 d) (41-43 d) (83-85 d) 1 Acetyltributylcitrat 98.4 83.8 60.6 2 Adimoll DB 97.5 90.8 75.5 3 Breakthru S240.sup.$ 61.3 38.5 20.2 4 Agnique AE829 95.3 89.4 71.9 5 Radia 7127 97.1 96 77.9 6 Radia 7208 92.6 91.7 75.9 7 Radia 7331 97.1 91.4 75.7 8 Radia 7368 98.1 91.6 74.4 9 Radia 7909 90.6 75.6 42.0 10 Radialube 7302 94.5 86.1 70.2 11 Triacetin 92.9 83.2 57.6 .sup.$= comparative example

[0151] Discussion: Spore viability directly after manufacturing of the samples (day 1) is generally high and at or above 90%. Examples according to the invention exhibit a spore viability of approx. 42% or greater after storage for approx. 3 months at 30° C. In many cases the fluids are exhibiting an even higher spore viability of approx. 70% or higher (Table IV, entries 2, 4-8, 10). For comparison, Break-Thru 5240 provides only 20% of spore viability after storage under conditions given here (Table IV entry 3).