NOVEL CARRIER FLUIDS FOR LIQUID FUNGAL SPORE FORMULATIONS

Abstract

The present invention relates to a liquid preparation comprising at least one ethoxylated and/or propoxylated organic liquid 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 essentially free of water comprising at least one ethoxylated and/or propoxylated organic liquid which is selected from the group consisting of a) ethoxylated fatty acid triglycerides with 3-10 ethylene oxide units wherein the fatty acid triglycerides are selected from the group consisting of castor oil and plant oils; b) a block copolymer of the general formula
H—O—[CH2-CH2-O-]a1-[CH2-CH(CH3)-O]b-[CH2-CH2-O-]a2-H where a1 and a2 have independently from each other an average value of between 1 and 20 and b has an average value of between 15 and 35; c) a polymer of the general formula
X—O—[CH2-CH(CH3)-O]m-[CH2-CH2-O-]n-Y where X and Y are independently selected from hydrogen branched or linear alkyl with 1-24 carbon atoms, and branched or linear carbonyl with 2-24 carbon atoms, saturated or partially unsaturated, optionally carrying hydroxyl functionality; where m is an average number between 0 and 10; where n is an average number between 0 and 40, where m+n is not zero or a mixture of any one of a) to c); and fungal spores.

2. The liquid preparation according to claim 1, wherein said ethoxylated fatty acid triglycerides according to a) are plant oils selected from the group consisting of sunflower oil, rapeseed oil, soybean oil, corn oil, coconut oil, and palm oil.

3. The liquid preparation according to claim 1, wherein said ethoxylated fatty acid triglycerides according to a) are castor oils.

4. The liquid preparation according to claim 1, wherein said ethoxylated and propoxylated organic liquid according to b) is selected from the group consisting of Block-Copolymers with an average mol wt. of between about 1000 and about 3000 g/mol and where a1 and a2 have independently from each other an average value of between 1 and 20 and b has an average value of between 15 and 35.

5. The liquid preparation according to claim 1, wherein said ethoxylated and propoxylated organic liquid according to b) is selected from the group consisting of Block-Copolymers with an average mol wt. of between about 1500 and about 3000 g/mol and where a1 and a2 have independently from each other an average value of between 10 and 15 and b has an average value of between 20 and 30.

6. The liquid preparation according to claim 1, wherein said ethoxylated and propoxylated organic liquid according to b) is selected from the group consisting of Block-Copolymers with an average molecular weight between about 2000 and about 3000 g/mol and where a1 and a2 have independently from each other an average value of between 3 and 16 and b has an average value of between 25 and 35; and Block-Copolymers with an average molecular weight between about 1400 and about 2200 g/mol and where a1 and a2 have independently from each other an average value of between 2 and 12 and b has an average value of between 15 and 25.

7. The liquid preparation according to claim 1, wherein in the polymer of c), X is branched or linear alkyl with 1-18 carbon atoms or branched or linear carbonyl with 2-18 carbon atoms, saturated or partially unsaturated, optionally carrying hydroxyl functionality and Y is hydrogen or branched or linear alkyl with 1-6 carbon atoms or branched or linear carbonyl with 2-6 carbon atoms, saturated or partially unsaturated, optionally carrying hydroxyl functionality, or wherein in the polymer of c), X is branched or linear alkyl with 1-6 carbon atoms or branched or linear carbonyl with 1-6 carbon atoms, saturated or partially unsaturated, optionally carrying hydroxyl functionality and y is branched or linear alkyl with 1-18 carbon atoms or branched or linear carbonyl with 2-18 carbon atoms, saturated or partially unsaturated, optionally carrying hydroxyl functionality.

8. (canceled)

9. The liquid preparation according to claim 1, wherein in the polymer of c) m+n is between 1 and 30.

10-11. (canceled)

12. The liquid preparation according to claim 1, wherein, in the polymer of c), if m equals zero the molecular weight (MW) of said at least one ethoxylated organic liquid is greater than or equal to 190 mass units.

13. The liquid preparation according to claim 12, where if m equals zero the molecular weight (MW) of said at least one ethoxylated organic liquid is greater than or equal to 205 mass units.

14. The liquid preparation according to claim 1, wherein said ethoxylated and/or propoxylated organic liquid according to c) is selected from the group consisting of polyethylene oxide, ethoxylated alcohols, mono-/polyethylene oxide diethers, mono-/polyethylene oxide ether-ester, ethoxylated carboxylic acids, mono-/polyethylene oxide di-esters, polypropylene oxide, propoxylated alcohols, mono-/polypropylene oxide diethers, mono-/polypropylene oxide ether-ester, propoxylated carboxylic acids, mono-/polypropylene oxide di-esters, alcohol propoxylate-ethoxylates, carboxylic acid propoxylate-ethoxylates and carboxylic acid propoxylate-ethoxylate ethers.

15. The liquid preparation according to claim 1, wherein said ethoxylated and/or propoxylated organic liquid is present in an amount of at least 40 wt.-%, preferably at least 50 wt.-%.

16. The liquid preparation according to claim 1, which is water-miscible.

17-19. (canceled)

20. The liquid preparation according to claim 1, wherein said fungal species is selected from the group consisting of Isaria fumosorosea, Penicillium bilaii, Metarhizium anisopliae, Purpureocillium lilacinum, Coniothyrium minitans, Beauveria bassiana and Clonostachys rosea.

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

22. The liquid preparation according to claim 21, wherein said rheology modifier is selected from the group consisting of fumed (hydrophobic/hydrophilic) or precipitated silica, silica of natural origin, attapulgite-based rheology modifiers, organo-modified clays, and mixtures thereof.

23. The liquid preparation according to claim 22, wherein said rheology modifier is fumed silica.

24. The liquid preparation according to any one of the preceding claims, comprising 0.1 to 40 wt.-% of fungal spores, up to 99.9% of at least one ethoxylated and/or propoxylated organic liquid according to any one of claims 1 to 16 and 0 to 10 wt.-% of at least one surfactant and/or rheology modifier.

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

26. 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 any of the preceding claims to said plant or to a locus where plants are growing or intended to be grown.

27. (canceled)

Description

EXAMPLE I (P. LILACINUM)

[0128] 3 g of P. lilacinum strain 251 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.

[0129] 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 2504 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 is used for further dilution and spotting on agar.

[0130] 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 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 25° C. for 17 hours. Open the plate and place it under the microscope. Randomly choose 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.

[0131] The results of spore viabilities are given in table I.

TABLE-US-00003 TABLE I spore viability [%] after after after after after after after at 2 w 1 m 5 w 6 w 2 m 3 m 7 m No Liquid day 1 (12-15 d) (27-28 d) (35 d) (40 d) (51-55 d) (87-92 d) (210 d) 1 Atlas G5002L * 97.4 96.0 90.7 2 Atplus 245 64.3 56.6 58.1 44.1 3 Berol 050 96.1 89.1 92.7 66.9 59.1 4 Berol 260 79.0 83.7 83.9 73.7 5 Butylcarbitol# 92.7 62.1 10.8 3.2 0.6 6 Butylcellosolve# 68.1 35.8 22.4 7 Dipropylene glycol 95.0 94.5 85.4 8 Dowanol DPM 91.0 95.3 93.3 74.7 9 Dowanol DPMA 99.1 93.0 35.7 10 Dowanol PGDA 98.8 98.2 88.4 11 Dowanol TPM 85.4 94.3 95.6 84.5 12 Ecosurf EH3 93.1 80.1 64.4 13 Etocas 10 * 97.0 97.2 97.8 14 Hexylcellosolve# 79.5 27.4 6.0 15 Leofat OC0503M 96.2 96.0 93.5 16 Lucramul CO08 98.7 91.9 86.3 17 Lucramul HOT 5902 86.5 86.0 66.2 18 Lucramul L03 99.0 90.1 86.3 19 Lucramul L05 97.5 89.2 71.2 20 Methoxytriglycol# 83.3 55.3 19.9 21 n-Butyldiglycolacetat 93.2 89.3 82.7 22 Proglyde DMM 98.8 96.0 75.4 23 Propylcellosolve# 76.2 8.1 0.6 24 Radiasurf 7402 96.7 83.6 64.8 34.7 25 Radiasurf 7403 98.6 98.3 98.9 93.7 78.3 26 Radiasurf 7423 90.8 91.4 93.8 79.7 36.9 27 Radiasurf 7442 98.7 96.5 98.2 96.8 88.3 28 Synperonic PE/L 44 94.5 94.2 53.4 29 Synperonic PE/L 62 97.1 96.9 95.3 91.6 61.4 30 Synperonic PE/L 64 96.5 92.3 64.8 31 Triethylenglycol 77.5 57.5 50.8 monobutylether 32 Tetraglyme 97.2 97.6 94.8 93.2 83.5 33 Breakthru S240$ 90.5 91.8 85.8 76.8 37.3 7.4 * Sample could not be evaluated for technical reasons; $control, average values out of 6 trials; #not according to the invention.

Discussion:

[0132] From the results depicted in Table I it becomes evident that not every fluid is suitable to provide good spore viabilities after storage. Spore viability directly after manufacturing of the samples (day 1) is generally high and in most cases at or above 80%, in many cases even above 90%. Certain fluids that do not belong to the scope of this invention exhibit a steep decline in spore viability even at day 1 after preparation of the samples and even more after storage under given conditions (Table I, entries 5, 6, 14, 20, 23). BreakThru 5240 has been previously described as a superior fluid to host fungals spores. Under the given test conditions BreakThru 5240 (Table I, entry 33) provides ˜77% spore viability after 2 m of storage and ˜7% after 7 m of storage. Among the examples according to the invention are selected fluids that exhibit a spore viability of approx. 53% or greater after storage for 2 or 3 months, respectively. In many cases, spore viabilities of approx. 80% or greater were detected (Table I, entries 1, 7, 10, 11, 13, 15, 16, 18, 21, 26, 29). For selected fluids exceptional spore viability was found even after storage for 7 months at 30° C. (Table I, entries 25, 27, 32).

EXAMPLE II (ISARIA FUMOSOROSEA)

[0133] Method 1: 1.5 g of Isaria 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.

[0134] 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 2504 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 is used for further dilution and spotting on agar.

[0135] For evaluation of spore germination rate a 1:15000 dilution based on the 1:100 dilution achieved by multiple automated dilution (pipetting robot, 96 well plate) was prepared. Afterwards 12×12 cm agar plates were taken and spotted with 10 times 5 μL of each sample using an automated 12-Channel pipet. After all liquid is soaked up by agar the agar plates were incubated at 23° C. for 16 hours.

[0136] Method 2: 0.5 g of Isaria fumosoresea pure spore powder were transferred into an Erlenmeyer flask using a sterile spoon. 24.5 mL of fluid were dispersed using Ultra-Turrax (IKA; Type T 25 D) for 1 min at 3,000 rpm. After this, 2.0 mL were transferred in 20 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.

[0137] 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 0.5 g of each sample were transferred into 100 mL-Erlenmeyer flask. The flasks were filled up to 50 g using a sterile aqueous solution containing 0.1% Break Thru® 5240 (Evonik, Industries) and homogenized on a magnetic stirrer (Thermo Fisher Scientific: Cimarec™) at least for 15 min with 750 rpm to achieve the first dilution step (1:100 dilution). This dilution was used for further dilution.

[0138] Not all samples mixed well or mixed at all in 0.1% Break Thru 5240. For these samples, 500 μL Neo-wett (Kwizda Agro GmbH) and/or a small amount (knife tip) Metaupon®-OMT (LEUNA Tenside GmbH) were also added to the aqueous solution.

[0139] After homogenization 1 mL were transferred into a 1.5 mL tube and were centrifuged for 15 min at 14,000 rpm (Thermo Fisher Scientific, Type Megafuge 8R). The supernatant (=upper phase) was discarded by using a pipette. The tubes were filled up to 1 mL using a sterile aqueous solution containing 0.1% Neo-wett and homogenized by vortexing.

[0140] To assess fungal viability, a method based on flow cytometry was used.

[0141] The results of spore viabilities are given in table II.

TABLE-US-00004 TABLE II spore viability [%] at after 2 w after 1 m after 7 m No Liquid day 1 (~14 d) (27 d) (210 d) Method 1 Butylcarbitol.sup.# 19.1 0.5 2.4 1 2 Carbitol.sup.# 78.0 13.7 12.0 2 3 Alkamuls A 94.4 93.0 90.7 2 4 Ariatone TV 97.0 71.7 66.7 2 5 Dowanol DPMA 87.7 63.6 31.4 1 6 Dowanol TPM 88.0 65.4 52.6 1  6a Dowanol TPM 82.0 59.0 2 7 Etocas 10 93.7 82.9 75.1 1  7a Etocas 10 93.8 92.0 88.8 2 8 Lucramul HOT 5902 93.0 32.0 2 9 Lutensol AO3 95.8 93.6 84.6 2 10  Lutensol AO7 94.4 92.8 82.9 2 11  Pluriol E300 96.0 87.0 45.0* 2 12  Propylenglycol Diacetat 97.0 53.0 2 13  Radiasurf 7402 92.5 11.6 1.1 1 13a Radiasurf 7402 89.9 20.5 15.8 2 14  Radiasurf 7403 94.0 76.2 79.3 1 14a Radiasurf 7403 89.0 86.0 92.0 2 15  Radiasurf 7423 86.7 58.0 42.3 2 16  Radiasurf 7442 89.4 72.1 58.1 1 16a Radiasurf 7442 87.4 69.6 66.3 2 17  Tween 20 98.6 98.9 (98.1**) 2 18  Tween 80 96.2 76.4 72.0 2 19  Tween 85 98.9 98.0 96.1*** 2 20  Synperonic PE/L 62 91.6 72.8 74.9 1 21  Breakthru S240.sup.$ 90.9 78.7 69.6 1 spore viability of I. Fumosorosea after storage; .sup.#not according to the invention; .sup.$control; *after 3 months; **after storage for 2 weeks at 40° C., ***after 6 months

Discussion:

[0142] Spore viability directly after manufacturing of the samples (day 1) is generally high and in most cases at or above 90% with the exception of Butylcarbitol and Carbitol (Table II, entry 1, 2) which were used as negative standard (i.e. not according to the invention). As demonstrated with a few examples (Table 2, entries 6/6a, 7/7a, 13/13a, 14/14a, 16/16a) both methods for determination of spore viability provide comparable results. Among the examples according to the invention are selected fluids that exhibit a spore viability of approx. 60% or greater after storage for 1 month (Table II, entries 3-7, 9-12, 14, 16,-1-20) or approx. 50% or greater after storage for 7 m at 30° C. (Table 2 entries 6, 7, 14, 16, 19, 20).

EXAMPLE III (BEAUVERIA BASSIANA)

[0143] 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.

[0144] 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 2504 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 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 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 choose 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 3 w after 5 w No Liquid 1 (14 d) (21 d) (35 d) 1 Atlas G5002L 91.9 72.4 2 Berol 050 88.7 78.7 3 Berol 260 86.0 72.4 4 Butylcarbitol 53.3 0.8 5 Dipropylene glycol 64.0 52.8 51.7 6 Proglyde DMM 88.4 48.0 21.8 7 Dowanol DPM 83.9 61.0 36.6 8 Dowanol TPM 63.9 19.0 15.5 9 Etocas 10 90.6 81.5 10 Leofat OC0503M 88.1 82.5 11 Lucramul HOT 5902 80.1 71.1 12 Methoxytriglycol 79.7 12.3 1.5 13 Dowanol PGDA 90.6 51.6 14 Radiasurf 7403 81.4 77.3 72.3 15 Radiasurf 7442 85.9 33.2 16 Synperonic PE/L 62 89.0 66.1 17 Synperonic PE/L 64 87.8 70.0 # not according to the invention

Discussion:

[0146] Spore viability directly after manufacturing of the samples (day 1) is generally high and in most cases at or above 80%, in many cases even above 90%. Fluids that do not fall within the scope of this invention exhibit a steep decline in spore viability after storage under given conditions (Table III, entries 4, 12). Among the examples according to the invention are selected fluids that exhibit a spore viability of approx. 50% or greater after storage for 3w or longer at 30° C. (Table III, entries 1, 2, 3, 5, 9, 10, 14, 17)

EXAMPLE IV: PENICILLIUM BILAII

[0147] 1.5 g of Penicillium bilaii (ATCC 20851) 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 2504 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 is used for further dilution and spotting on agar.

[0149] 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 choose 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 Atlas G5002L 95.1 76.3 55.8 2 Berol 050 65.7 50.9 37.7 3 Berol 260 51.4 27.2 20 4 Breakthru S240# 53.1 27.6 19.6 5 Dipropylene glycol 83.8 44.1 29.5 6 Dipropylene glycol DME 54.6 29.6 10.5 7 Dowanol PGDA 93.6 69.3 36.4 8 Dowanol TPM 67.2 7.6 2.8 9 Etocas 10 97.2 78.8 65.9 10 Leofat OC0503M 93.4 73.3 64.1 11 Lucramul HOT 5902 45.8 27.8 20 12 Methoxytriglycol 74.3 43.1 30.3 13 Radiasurf 7355 * 78.9 40.4 14 Radiasurf 7403 78.1 66.8 61.8 15 Radiasurf 7442 96.7 91 76.5 16 Synperonic PE/L 62 98.4 84.1 61.4 17 Synperonic PE/L 64 83.5 68 51.3 18 Tween 85 99.0 92.0$ * Sample could not be evaluated for technical reasons; #control; $after 2 months

Discussion:

[0150] Spore viability directly after manufacturing of the samples (day 1) is quite variable; in most cases the viability is at or above 50%, in selected cases even above 90%. Among the examples according to the invention are selected fluids that exhibit a spore viability of approx. 50% or greater after storage for approx. 3 months at 30° C. (Table IV, entries 1, 10,11,15-17). In many cases the spore viability in selected fluids is above that of Break-Thru 5240 used as control after storage for approx. 3 months at 30° C., i.e. above approx. 20%. Some fluids provide inferior spore viability under conditions given here.