NOVEL FORMULATIONS FOR INCREASING THE GERMINATION RATE OF FUNGAL SPORES

Abstract

The present invention relates to a preparation for stabilizing fungal spores. The formulation comprises at least one glycerophospholipid and spores of a fungal microorganism.

Claims

1. A composition of matter, which is (a) a preparation comprising at least one glycerophospholipid and spores of a fungal microorganism in a weight ratio range of between 10:1 and 1:5000; or (b) a liquid composition comprising the preparation according to (a) and at least one carrier.

2. The preparation according to claim 1(a), wherein said glycerophospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid and mixtures of any of the foregoing.

3. The preparation according to claim 1(a), wherein said weight ratio range is between 2:1 and 1:5.

4. The preparation according to claim 1(a), wherein said glycerophospholipid is a lecithin.

5. The preparation according to claim 1(a), wherein said fungal spores are of a fungal species selected from the group consisting of Trichoderma spp., Isaria spp., Beauveria spp., Paecilomyces spp., Coniothyrium spp.

6. The preparation according to claim 1(a), wherein said fungal spores are of Trichoderma spp.

7. The preparation according to claim 5, wherein said Trichoderma spp. is selected from Trichoderma harzianum, Trichoderma viride, Trichoderma atroviride, Trichoderma asperellum, Trichoderma virens, Trichoderma gamsii, Trichoderma polysporum, Trichoderma stromaticum and Trichoderma koningii.

8. The preparation according to claim 1(a), wherein said preparation does not contain vegetable oil.

9. (canceled)

10. The composition according to claim 1(b), wherein said at least one carrier is selected form the group consisting of plant oils, polyether-modified trisiloxanes, carboxylic esters and an ethoxylated and/or propoxylated organic liquid.

11. The composition according to claim 1(b), wherein said at least one carrier is Radiasurf 7403, Radiasurf 7442, Triton X 100, PEG300, triacetin, Atlas G5002 and/or Tween20.

12. The composition according to claim 1(b), further comprising a rheology-modifying agent.

13. The composition according to claim 12, wherein said rheology-modifying agent is fumed or precipitated silica, preferably Aerosil 200.

14. The composition according to claim 1(b) which is a seed treatment composition.

15. A seed coated with the liquid composition according to claim 1(b).

16. The seed according to claim 15, further comprising at least one plant protection agent.

17. The seed according to claim 16, wherein said at least one plant protection agent is selected from the group consisting of prothioconazole, metalaxyl, mefenoxam, fluoxastrobin, tebuconazole, ipconazole, metconazole, cyproconazole, epoxiconazole, propiconazole, azoxystrobin, pyraclostrobin, picoxystrobin, benzovindiflupyr, fluxapyroxad and chlorothalonil.

18. Method of (a) producing a preparation according to claim 1(a), comprising the steps of providing non-dried fungal spores, adding at least at least one glycerophospholipid in a weight ratio of between 10:1 and 1:5000 and drying the resulting mixture; or (b) producing a liquid composition according to claim 1(b), comprising the steps of providing non-dried fungal spores, adding at least one glycerophospholipid in a weight ratio of between 10:1 and 1:5000, drying the resulting mixture and mixing it with at least one carrier; or c) increasing the germination rate of spores of a fungal microorganism comprising the steps of providing non-dried fungal spores, adding at least one glycerophospholipid in a weight ratio of between 10:1 and 1:5000, drying the resulting mixture and mixing it with at least one carrier; or (d) controlling phytopathogenic fungi, insects, spiders, molluscs, weeds, rodents and/or nematodes in, on or around a plant or plant part, for enhancing growth of a plant or for increasing plant yield or root health comprising applying the composition of matter of claim 1 to said plant or plant part or to a plot where plants are to be grown; or (e) stabilizing spores of fungal microorganisms, preferably those obtained with wet-harvest, or increasing the germination rate of spores of fungal microorganisms, the method comprising applying at least one glycerophospholipid.

19. (canceled)

20. (canceled)

21. The method of claim 18(a), wherein said drying is effected as air-drying or spray-drying or vacuum drum drying.

22. (canceled)

23. (canceled)

24. (canceled)

25. (canceled)

26. The method of claim 18(d), wherein said composition of matter is applied to seed.

Description

EXAMPLE 1: COMPARISON OF DIFFERENT PROTECTANT FOR TRICHODERMA SPP. STRAINS

[0244] A test was conducted to compare lecithin against other known microbial stabilizers and protectant materials. These materials include different sugars, proteins, skim milk powder, and others. Initially, an air-drying technique in room temperature was used.

[0245] After the spores were air dried with different protectants at room temperature overnight, the germination rate and total dry matter content (DMC, which is a way of expressing total moisture content) of the samples were analyzed. Table 1 shows the results for two strains of Trichoderma.

TABLE-US-00004 TABLE 1 Comparison of Trichoderma germination rates after air-drying with no protectants, with conventional and theoretical stabilizers and protectants (1:1 material:spore) vs. with lecithin (1:10 material:spore). Germination rate (%) DMC (%) T1 B35 T1 B35 no protectants 62.21 74.63 91.05 90.04 Yeast without AA 15.54 4.69 91.79 92.52 Amino Acids Skim milk powder 67.34 25.84 89.63 90.46 Maltodextrin 54.55 20.38 94.46 92.49 Chitosan not soluble — — Proteinshake 41.58 17.76 92.52 93.02 Lecithin 74.63 73.56 95.43 92.88 Sucrose spores not 36.45 — 91.59 processable Trehalose 68.75 43.18 90.35 91.84 Sorbitol/Glucitol spores not 13.00 — 93.99 processable Ducitol/Galactitol not soluble — — Mannitol 69.95 51.40 97.01 94.88 Myo-Inositol 65.03 59.05 96.6 95.59

[0246] As shown in Table 1, lecithin has shown superior performance in terms of maintaining the germination rate of Trichoderma strains over other tested materials in these tested conditions. In our example, the superiority of lecithin over other materials tested is more evident in the case of the more inherently sensitive strain variety B35 (Trichoderma asperellum). It is also noteworthy that Lecithin-to-spore ratio in these experiments were 1:10, while the other commonly known or theoretical stabilizers and protectants were used in material-to-spore ratio of 1:1. In other words, lecithin was used in 10 times less concentration, compared to other materials tested. These superior effects seen from lecithin are observed in this test with 10 times less concentrations compared to other fungal stabilizer and protectant materials used.

EXAMPLE 2: COMPARISON OF PROTECTANTS AT INDUSTRIAL SCALE

[0247] At the next stage in evaluation, we wanted to also show these superior results in an example of industrially-scalable technique, such as spray-drying for example. A pilot-size spray-dryer equipment was used, as described above. Using such instrument, the effect of lecithin on the viability of two exemplary Trichoderma strain (in this case T1) was evaluated.

[0248] Tables 2 and 3 show the results and parameters used in spray-drying of the tested Trichoderma strains without stabilizer or protectant, with lecithin, and with two of the most common protectants (trehalose, and skim milk powder) used for microorganisms. The results for this example show a benefit ranging from 40-100% increase in germination rate after spray-drying with lecithin as opposed to when no protectant or stabilizer is added. More importantly, in comparison with two other widely-used protectants (trehalose and skim milk powder), it surely shows outperformance. This is in alignment with our previous experiment in example 1 in which lecithin also outperformed in increasing the germination rate of the dried spores.

TABLE-US-00005 TABLE 2 Comparison of Trichoderma germination rates in a pilot-scale spray-dryer (in presence and absence of lecithin/in comparison with other protectants). In this study protectant-to-spore mass ratio was selected to be 1:1. Rotating Residual Viability Solids conc Speed Temp Temp Product Product in moisture Feed Viability normalized mean in feed Atomizer Inlet Outlet in Filter Cyclone Cyclone before drying after drying w % U/min ° C. ° C. — — % % % Trichoderma (Paste) + 5% 5 15000 45 30 0% 100% 7.2 99 79.5 lecithin Trichoderma (Paste) + 5% 5 15000 45 30 0% 100% 5.67 99 78.5 lecithin + 5% Trehalose Trichoderma (Paste) 5 15000 45 30 0% 100% 7.72 99 39.3 Trichoderma (Paste) + 5% 5 15000 45 30 0% 100% 7.31 99 56.5 Trehalose Trichoderma (Paste) + 5% 5 15000 45 30 0% 100% 6.6 99 62.1 Skim milk powder

TABLE-US-00006 TABLE 3 Repeating comparison of Trichoderma germination rates in a pilot-scale spray-dryer (in presence and absence of lecithin). In this study protectant- to-spore mass ratio was selected to be 1:1. (Values in bracket: new determination of viability after shipment of material under unknown conditions) Rotating Residual Viability Solids conc Process Speed Temp Temp Product Product in moisture Feed Viability normalized mean in feed Time Atomizer Inlet Outlet in Filter Cyclone Cyclone before drying after drying w % min U/min ° C. ° C. — — % % % Trichoderma (Paste) 3.76 62 25000 50 30 0% 100% 7.8 97.3 56.9 Trichoderma (Paste) 8.12 33 25000 50 30 0% 100% 6.82 97.3 55.6 Trichoderma (Paste) 3.6 95 15000 45 30 0% 100% 7 97.5 50.7 (41.8) Trichoderma (Paste) + 11.3 142 15000 45 30 0% 100% 4.2 97.5 68.9 (62.1) 5% lecithin

[0249] The spore powders a) without lecithin and a germination rate of 41.8% and the spore powder b) with lecithin (ratio 1:1) and a germination rate of 62.1% were studied during storage at 4° C. and minus 20° C. The powders were stored in closed plastic tubes and the germination rate was analyzed overtime. Lecithin showed a significant protecting effect after drying of the spores (t0), and a stabilizing effect during storage at minus 20° C. (see table 4).

TABLE-US-00007 TABLE 4 Germination rate over time of TI Trichoderma spore powder spray-dried with and without lecithin. Storage conditions are 4° C. and −20° C. 4° C. −20° C. Storage powder powder powder powder time without with without with (months) lecithin lecithin lecithin lecithin 0 41.8 62.1 41.8 62.1 0.5 31.0 45.4 29.7 53.9 1 39.5 42.5 20.9 38.0 2 41.9 43.2 22.3 51.5 3 27.7 31.8 26.6 55.2 6 21.6 24.3 30.7 59.2

EXAMPLE 3: GERMINATION RATE OF FORMULATED FUNGAL SPORES

[0250] The next stage of evaluation in this example is to determine the germination rate of spores treated with lecithin and disbursed in liquid carriers and formulations. For this reason, we selected few different example carriers and made formulations of T1 Trichoderma (both spray-dried and spray-dried with lecithin) with and without silica (Aerosil 200 from Evonik) as rheology-modifier. Such formulations were stored at different storage temperatures (20, 25, and 30° C., respectively) under Nitrogen. The germination rate of the spores in each formulation was measured over time. Tables 5-7 show the results for the samples with no silica, and Tables 8-10 show the results for samples with silica, as rheology modifier. Both groups indicate the outperformance of lecithin in combination with some liquid carriers.

TABLE-US-00008 TABLE 5 Comparison between germination rate of T1 Trichoderma spray-dried with and without lecithin and then dispersed into different formulation carriers (no silica added). Storage condition is 20° C. without 2% Aerosil (AE200) 20° C. BreakThruS240 Radiasurf 7403 Triacetin Atlas G5002L Spray- Spray- Spray- Spray- Spray- Spray- Spray- dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 storage without with without with without with without time protectant lecithin protectant lecithin protectant lecithin protectant (months) Germination Rate (%) 0 41.81 62.11 41.81 62.11 41.81 62.11 41.81 0.5 27.10 57.51 37.34 59.03 8.24 20.02 43.31 1 16.74 36.57 27.87 56.68 6.27 13.76 24.36 1.5 17.65 37.36 25.66 58.78 9.09 9.09 26.86 3 7.25 35.71 23.96 53.03 3.32 10.8 23.97 15 5.48 8.59 5.48 24.01 0.99 2.91 7.41 20° C. Atlas G5002L Etocas 10 DEVTween 20 AgniqueAE829 Spray- Spray- Spray- Spray- Spray- Spray- Spray- dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 storage with without with without with without with time lecithin protectant lecithin protectant lecithin protectant lecithin (months) Germination Rate (%) 0 62.11 41.81 62.11 41.81 62.11 41.81 62.11 0.5 53.80 30.94 48.79 13.33 67.42 16.34 38.67 1 50.86 16.16 47.67 19.14 57.53 24.38 43.82 1.5 51.66 22.51 44.86 20.07 49.08 18.18 36.37 3 39.22 9.39 45.63 13.13 42.53 13.99 10.45 15 22.72 4.46 12.28 7.41 25.56 0 4.03

TABLE-US-00009 TABLE 6 Comparison between germination rate of T1 Trichoderma spray-dried with and without lecithin and then dispersed into different formulation carriers (no silica added). Storage condition is 25° C. without 2% Aerosil 200 (AE200) 25° C. BreakThruS240 Radiasurf 7403 Triacetin Atlas G5002L Spray- Spray- Spray- Spray- Spray- Spray- Spray- dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 storage without with without with without with without time protectant lecithin protectant lecithin protectant lecithin protectant (months) Germination Rate (%) 0 41.81 62.11 41.81 62.11 41.81 62.11 41.81 0.5 26.68 47.22 33.53 63.64 7.09 16.97 36.43 1 17.36 46.93 24.77 69.09 7.31 15.65 34.59 25° C. Atlas G5002L Etocas 10 DEVTween 20 AgniqueAE829 Spray- Spray- Spray- Spray- Spray- Spray- Spray- dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 storage with without with without with without with time lecithin protectant lecithin protectant lecithin protectant lecithin (months) Germination Rate (%) 0 62.11 41.81 62.11 41.81 62.11 41.81 62.11 0.5 63.26 27.08 59.39 28.19 69.18 15.86 45.02 1 60.17 21.84 46.29 23.98 52.65 15.64 32.30

TABLE-US-00010 TABLE 7 Comparison between germination rate of T1 Trichoderma spray-dried with and without lecithin and then dispersed into different formulation carriers (no silica added). Storage condition is 30° C. without 2% Aerosil 200 (AE200) 30° C. BreakThruS240 Radiasurf 7403 Triacetin Atlas G5002L Spray- Spray- Spray- Spray- Spray- Spray- Spray- dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 storage without with without with without with without time protectant lecithin protectant lecithin protectant lecithin protectant (months) Germination Rate (%) 0 41.81 62.11 41.81 62.11 41.81 62.11 41.81 0.5 45.02 57.73 38.71 74.58 11.88 23.32 59.85 30° C. Atlas G5002L Etocas 10 DEVTween 20 AgniqueAE829 Spray- Spray- Spray- Spray- Spray- Spray- Spray- dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 storage with without with without with without with time lecithin protectant lecithin protectant lecithin protectant lecithin (months) Germination Rate (%) 0 62.11 41.81 62.11 41.81 62.11 41.81 62.11 0.5 77.84 36.03 79.16 37.02 73.86 30.89 46.89

TABLE-US-00011 TABLE 8 Comparison between germination rate of T1 Trichoderma spray-dried with and without lecithin and then dispersed into different formulation carriers (Aerosil 200 added). Storage condition is 20° C. with 2% Aerosil 200 (AE200) 20° C. Radiasurf Atlas BreakThru245 7403 + AE200 Triacetin + AE200 G5002L + AE200 Spray- Spray- Spray- Spray- Spray- Spray- Spray- dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 storage without with without with without with without time protectant lecithin protectant lecithin protectant lecithin protectant (months) Germination Rate (%) 0 41.81 62.11 41.81 62.11 41.81 62.11 41.81 0.5 36.96 66.27 36.46 0?  12.89 34.04 35.64 1 23.17 52.79 25.40 59.05 8.88 21.03 23.11 1.5 21.90 42.09 26.57 59.88 9.24 13.06 25.07 3 13.11 32.74 18.44 49.35 3.38 27.13 17.78 15 5.48 15.25 5.48 23.66 3.10 8.93 7.41 20° C. Atlas DEVTween G5002L + AE200 Etocas 10 + AE200 20 + AE200 AgniqueAE829 + AE200 Spray- Spray- Spray- Spray- Spray- Spray- Spray- dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 storage with without with without with without with time lecithin protectant lecithin protectant lecithin protectant lecithin (months) Germination Rate (%) 0 62.11 41.81 62.11 41.81 62.11 41.81 62.11 0.5 71.72 30.42 57.03 25.49 62.78 38.85 35.65 1 48.10 28.82 45.26 21.08 54.59 28.61 39.40 1.5 53.17 23.99 46.47 20.45 49.78 21.69 28.29 3 44.9 13.68 33.72 9.25 41.21 18.93 12.11 15 27.01 2.72 12.13 3.54 21.47 2.15 8.93

TABLE-US-00012 TABLE 9 Comparison between germination rate of T1 Trichoderma spray-dried with and without lecithin and then dispersed into different formulation carriers (Aerosil 200 added). Storage condition is 25° C. with 2% Aerosil 200 (AE200) 25° C. Radiasurf Atlas BreakThru245 7403 + AE200 Triacetin + AE200 G5002L + AE200 Spray- Spray- Spray- Spray- Spray- Spray- Spray- dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 storage without with without with without with without time protectant lecithin protectant lecithin protectant lecithin protectant (months) Germination Rate (%) 0 41.81 62.11 41.81 62.11 41.81 62.11 41.81 0.5 34.48 54.81 30.76 67.74 17.67 37.07 39.48 1 25.56 51.10 23.33 65.86 10.39 23.61 28.83 25° C. Atlas DEVTween G5002L + AE200 Etocas 10 + AE200 20 + AE200 AgniqueAE829 + AE200 Spray- Spray- Spray- Spray- Spray- Spray- Spray- dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 storage with without with without with without with time lecithin protectant lecithin protectant lecithin protectant lecithin (months) Germination Rate (%) 0 62.11 41.81 62.11 41.81 62.11 41.81 62.11 0.5 68.10 27.00 64.48 25.90 67.75 20.79 44.00 1 62.00 16.76 49.17 20.32 61.25 31.28 27.45

TABLE-US-00013 TABLE 10 Comparison between germination rate of T1 Trichoderma spray-dried with and without lecithin and then dispersed into different formulation carriers (Aerosil 200 added). Storage condition is 30° C. with 2% Aerosil 200 (AE200) 30° C. BreakThruS240 Radiasurf 7403 Triacetin Atlas G5002L Spray- Spray- Spray- Spray- Spray- Spray- Spray- dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 storage without with without with without with without time protectant lecithin protectant lecithin protectant lecithin protectant (months) Germination Rate (%) 0 41.81 62.11 41.81 62.11 41.81 62.11 41.81 0.5 50.61 62.99 44.93 70.99 23.09 39.71 52.73 30° C. Atlas G5002L Etocas 10 DEVTween 20 AgniqueAE829 Spray- Spray- Spray- Spray- Spray- Spray- Spray- dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 dried T1 storage with without with without with without with time lecithin protectant lecithin protectant lecithin protectant lecithin (months) Germination Rate (%) 0 62.11 41.81 62.11 41.81 62.11 41.81 62.11 0.5 70.79 36.25 61.99 52.16 65.48 49.09 41.94

EXAMPLE 4: VACUUM DRUM DRYING OF THE SPORE PASTE WITH AND WITHOUT LECITHIN AND GERMINATION RATE OVER TIME/SHELF LIFE STABILITY OF THE PURE SPORE POWDER

[0251] In this example we wanted to test another drying method in industrial scale. The spore suspension is rinsed onto a heated drum in a vacuum chamber (2-5 mbar) and dried on the drum surface while rotating (0.65 rpm) and harvesting the dried spore powder into a bowl by a knife scraping on the rotating drum.

TABLE-US-00014 TABLE 11 Trichoderma germination rates in a pilot-scale vacuum-drum-dryer (in presence and absence of lecithin). In this study protectant-to-spore mass ratio was selected to be 1:1. Speed Solids adding Viability Viability conc in Process feed Temp Temp Product Residual before after feed Time suspension Drum Product output moisture drying drying w % min g/min ° C. ° C. g % % % Trichoderma 7.56 17 14 50 46-40 13.2 6.88 98.61 48.0 (Paste) Trichoderma 8.28 12 14 50 42-38 13.0 11.39 98.61 68.8 (Paste) + lecithin

[0252] The spore powders were stored in closed plastic tubes at 30° C. and the germination rate over time was studied. Lecithin showed a significant protecting effect for drying the spores, and a slight improving effect on the stability of the dried spores during storage.

TABLE-US-00015 TABLE 12 Germination rate over time of T1 Trichoderma spore powder vacuum-drum-dried with and without lecithin. Storage condition is 30° C. storage time Powder without Powder with (months) lecithin lecithin 0 48.00 68.80 1 13.98 28.28 2 9.91 15.25