METHOD FOR INCREASING STORAGE STABILITY OF FUNGAL SPORES

Abstract

The present invention relates to a method of producing fungal spores with prolonged storage stability or of prolonging the storage stability of fungal spores comprising subjecting fungal spores to a drying procedure to result in a residual moisture content of 12% or less and a composition comprising fungal spores obtained by said method.

Claims

1. A method of producing fungal spores with prolonged storage stability or of prolonging the storage stability of fungal spores comprising subjecting fungal spores to a drying procedure to result in a residual moisture content of less than 12%.

2. The method according to claim 1, wherein said residual moisture content is between 1.5 wt.-% and 6 wt.-%.

3. The method according to claim 1, wherein the germination rate of said spores after 6 months at room temperature is at least 80%.

4. The method according to claim 1, wherein said drying procedure comprises vacuum drying.

5. The method according to claim 4, wherein said vacuum drying is effected for up to 16 hours.

6. The method according to claim 4, wherein said vacuum drying is effected at a temperature of up to 30° C.

7. The method according to claim 4 wherein said vacuum drying is effected at a pressure of 10-1 mbar.

8. The method according to claim 1, wherein said fungal spores are conidia.

9. The method according to claim 1, wherein said fungal spores are from a fungus having beneficial activity in the area of plant protection.

10. The method according to claim 9, wherein said fungus has fungicidal, insecticidal, nematicidal or herbicidal activity or supports and/or promotes and/or stimulates plant health and/ or plant growth.

11. The method according to claim 9, wherein said fungus has fungicidal activity and is selected from the group consisting of Coniothyrium minitans, in particular strain CON/M/91-8 (Accession No. DSM-9660) (available as Contans ® from Prophyta, DE); Microsphaeropsis ochracea strain P130A (ATCC 74412); Aspergillus flavus, strain NRRL 21882 (available as Afla-Guard® from Syngenta) and strain AF36 (available as AF36 from Arizona Cotton Research and Protection Council, US); Gliocladium roseum, strain 321U from Adjuvants Plus; Talaromyces flavus, strain VII7b; Ampelomyces quisqualis, in particular strain AQ 10 (available as AQ 10® by IntrachemBio Italia); Gliocladium catenulatum (Synonym: Clonostachys rosea f. catenulate), in particular strain J1446 (e.g. Prestop® by Verdera Oy), strain IK726, strain 88-710 (WO2007/107000), strain CR7 (WO2015/035504), Trichoderma asperellum, in particular strain ICC 012 from Isagro or strain SKT-1 (e.g. ECO-HOPE® from Kumiai Chemical Industry), strain T34 (e.g. T34 Biocontrol by Biocontrol Technologies S.L., ES); Trichoderma viride, in particular strain TV1(e.g. Trianum-P by Koppert), strain B35 (Pietr et al., 1993, Zesz. Nauk. A R w Szczecinie 161: 125-137), Trichoderma atroviride, in particular strain CNCM 1-1237 (e.g. Esquive® WP from Agrauxine, FR), strain SC1 described in International Application No. PCT/IT2008/000196), strain 77B (T77 from Andermatt Biocontrol), strain no. V08/002387, strain NMI no. V08/002388, strain NMI no. V08/002389, strain NMI no. V08/002390, strain LC52 (e.g. Sentinel from Agrimm Technologies Limited), strain LUI32 (e.g. Tenet by Agrimm Technologies Limited), strain ATCC 20476 (IMI 206040), strain T11 (IM1352941/ CECT20498), strain SKT-1 (FERM P-16510), strain SKT-2 (FERM P-16511), strain SKT-3 (FERM P-17021) and Cladosporium cladosporioides, e.g. strain H39 (by Stichting Dienst Landbouwkundig Onderzoek).

12. The method according to claim 9, wherein said fungus has insecticidal activity and is selected from C2.3 Beauveria bassiana, in particular strain ATCC 74040; strain GHA (Accession No. ATCC74250); strain ATP02 (Accession No. DSM 24665); strain PPRI 5339; strain PPRI 7315, strain R444, strains IL197, IL12, IL236, IL10, IL131, IL116; strain BaGPK; strain ICPE 279, strain CG 716; C2.6 Lecanicillium lecanii (formerly known as Verticillium lecanii), in particular conidia of strain KV01, strain DAOM198499 or strain DAOM216596; C2.9 Lecanicillium muscarium (formerly Verticillium lecanii), in particular strain VE 6/CABI(=IMI) 268317/CBS102071/ARSEF5128; C2.10 Metarhizium acridum, e.g. ARSEF324 or isolate IMI 330189 (ARSEF7486); C2.11 Metarhizium brunneum, e.g. strain Cb 15 or strain F52 (DSM3884/ ATCC 90448); C2.12 Metarhizium anisopliae complex species/strains, e.g. strain ESALQ 1037, strain E-9, strain M206077, strain C4-B (NRRL 30905), strain ESC1, strain 15013-1 (NRRL 67073), strain 3213-1 (NRRL 67074), strain C20091, strain C20092, or strain ICIPE 78; C2.14 Paecilomyces fumosoroseus (new: Isaria fumosorosea), in particular strains Apopka 97, Fe9901, ARSEF 3581, ARSEF 3302, ARSEF 2679, IfB01 (China Center for Type Culture Collection CCTCC M2012400), ESALQ1296, ESALQ1364, ESALQ1409, CG1228, KCH J2, HIB-19, HIB-23, HIB-29, HIB-30, CHE-CNRCB 304, EH-511/3, CHE-CNRCB 303, CHE-CNRCB 305, CHE-CNRCB 307, EH-506/3, EH-503/3, EH-520/3, PFCAM, MBP, PSMB1, RCEF3304, PF01-N10 (CCTCC No. M207088), CCM 8367, SFP-198, K3, CLO 55, IfTS01, IfTS02, IfTS07, P1, If-02, If-2.3, If-03, Ifr AsC, PC-013 (DSMZ 26931), P43A, PCC, Pf04, Pf59, Pf109, FG340, Pfr1, Pfr8, Pfr9, Pfr10, Pfr11, Pfr12, Ifr531, IF-1106, I9602, I7284, I03011 (Patent U.S. Pat. No. 4,618,578), CNRCB1, SCAU-IFCF01, PF01-N4, Pfr-612, Pf-Tim, Pf-Tiz, Pf-Hal and Pf-Tic.

13. The method according to claim 9, wherein said fungus has nematicidal activity and is selected from D2.1 Muscodor albus, in particular strain QST 20799 (Accession No. NRRL 30547); D2.2 Muscodor roseus, in particular strain A3-5 (Accession No. NRRL 30548); D2.3 Paecilomyces lilacinus (also known as Purpureocillium lilacinum), in particular P. lilacinus strain 251 (AGAL 89/030550; e.g. BioAct from Bayer CropScience Biologics GmbH); D2.4 Trichoderma koningii; D2.5 Harposporium anguillullae; D2.6 Hirsutella minnesotensis; D2.7 Monacrosporium cionopagum; D2.8 Monacrosporium psychrophilum; D2.9 Myrothecium verrucaria, in particular strain AARC-0255 (e.g. DiTera™ by Valent Biosciences); D2.10 Paecilomyces variotii, strain Q-09 (e.g. Nemaquim® from Quimia, MX); D2.11 Stagonospora phaseoli (e.g. from Syngenta); D2.12 Trichoderma lignorum, in particular strain TL-0601 (e.g. Mycotric from Futureco Bioscience, ES); D2.13 Fusarium solani, strain Fs5; D2.14 Hirsutella rhossiliensis; D2.15 Monacrosporium drechsleri; D2.16 Monacrosporium gephyropagum; D2.17 Nematoctonus geogenius; D2.18 Nematoctonus leiosporus; D2.19 Neocosmospora vasinfecta; D2.20 Paraglomus sp, in particular Paraglomus brasilianum; D2.21 Pochonia chlamydosporia (also known as Vercillium chlamydosporium), in particular var. catenulata (IMI SD 187; e.g. KlamiC from The National Center of Animal and Plant Health (CENSA), CU); D2.22 Stagonospora heteroderae; D2.23 Meristacrum asterospermum, D2.24 Duddingtonia flagrans.

14. The method according to claim 9, wherein said fungus supports and/or promotes and/or stimulates plant health and plant growth and is E2.1 Talaromyces flavus, in particular strain V117b; E2.2 Trichoderma atroviride, in particular strain no. V08/002387, strain no. NMI No. V08/002388, strain no. NMI No. V08/002389, strain no. NMI No. V08/002390, strain LC52 (e.g. Sentinel from Agrimm Technologies Limited), strain kd (e.g. T-Gro from Andermatt Biocontrol), and/or strain LUI32 (e.g. Tenet from Agrimm Technologies Limited); E2.3 Trichoderma harzianum, in particular strain ITEM 908 or T-22 (e.g. Trianum-P from Koppert); E2.4 Myrothecium verrucaria, in particular strain AARC-0255 (e.g. DiTera™ from Valent Biosciences); E2.5 Penicillium bilaii, in particular strain ATCC 22348, and/or strain ATCC20851 (e.g. JumpStart® from Monsanto BioAg); E2.6 Pythium oligandrum, in particular strains DV74 or M1 (ATCC 38472; e.g. Polyversum from Bioprepraty, CZ); E2.7 Rhizopogon amylopogon (e.g. comprised in Myco-Sol from Helena Chemical Company); E2.8 Rhizopogon fulvigleba (e.g. comprised in Myco-Sol from Helena Chemical Company); E2.9 Trichoderma harzianum, in particular strain TSTh20, strain KD, product Eco-T from Plant Health Products, ZA or strain 1295-22; E2.10 Trichoderma koningii; E2.11 Glomus aggregatum; E2.12 Glomus clarum; E2.13 Glomus deserticola; E2.14 Glomus etunicatum; E2.15 Glomus intraradices; E2.16 Glomus monosporum; E2.17 Glomus mosseae; E2.18 Laccaria bicolor; E2.19 Rhizopogon luteolus; E2.20 Rhizopogon tinctorus; E2.21 Rhizopogon villosulus; E2.22 Scleroderma cepa; E2.23 Suillus granulatus; E2.24 Suillus punctatapies; E2.25 Trichoderma virens, in particular strain GL-21; and E2.26 Verticillium albo-atrum (formerly V. dahliae), in particular strain WCS850 (CBS 276.92; e.g. Dutch Trig from Tree Care Innovations).

15. The method of claim 1, wherein the fungal spores are from the species Metarhizium brunneum or Metarhizium acridum.

16. The method according to claim 15, wherein said fungal spores are from Metarhizium brunneun strain F52 or Metarhizium acridum strain ARSEF324.

17. The method according to claim 16, wherein the germination rate of said fungal spores after 6 months at room temperature is at least 85%.

18. The method according to claim 16, wherein the germination rate of said fungal spores after 8 months at room temperature is at least 80%.

19. The method of claim 16, wherein said drying is effected to a residual moisture content of between 1 and 5%.

20. The method of claim 1 wherein the fungal spores are from the species Isaria fumosorosea.

21. The method according to claim 20, wherein the fungal spores are from Isaria fumosorosea strain APOPKA97, Fe9901, ARSEF 3581, IfB01, ESALQ1296, ESALQ1364, ESALQ1409, CG1228, KCH J2, HIB-19, HIB-23, HIB-29, or HIB-30.

22. The method according to any one of claims 1 to 21, claim 1, further comprising packaging said spores under reduced oxygen and/or water exposure.

23. Fungal spores obtained by the method of claim 1.

24. A composition comprising the fungal spores according to claim 23.

25. A method of producing a composition, comprising formulating the fungal spores according to claim 23.

26. A method for controlling phytopathogenic fungi, insects, spiders, molluscs, weeds, rodents and/or nematodes in a plant, for enhancing growth of a plant or for increasing plant yield or root health, comprising applying fungal spores according to claim 23 to said plant or to a plot where plants are to be grown.

27. A method for controlling phytopathogenic fungi, insects, spiders, molluscs, weeds, rodents and/or nematodes in a plant, for enhancing growth of a plant or for increasing plant yield or root health, comprising applying a composition according to claim 24 to said plant or to a plot where plants are to be grown.

Description

[0060] FIGS. 1 and 2: Comparison of germination rate of Metarhizium brunneum spores fermented on two different substrates with different residual moisture contents

EXAMPLE 1

Drying Procedure for Fungal Spores

[0061] The conidia powder (before drying usually around 50% residual moisture) is evenly distributed in thermal conducting trays, making sure the layer of the powder is not thicker than 2 cm. The trays then are placed in the respective vacuum-dryer. After that a vacuum is created in the drying chamber, enabling the residual moisture to evaporate from the powder. The moisture is pumped out of the drying chamber by the vacuum pump and the powder subsequently dried.

[0062] The drying trays are heated to a set temperature which is constantly adjusted to account for evaporative cooling. A temperature sensor in the powder shows the change in the temperature of the powder. If this temperature increases towards the temperature of the tray it indicates that at the given vacuum there is only little moisture left that will evaporate from the powder. Then a greater (bigger/stronger) vacuum can be created to enable more moisture to leave the powder or the powder reached the desired moisture level. The moisture level is determined by stopping the drying process, breaking the vacuum, taking a small, representative sample and analyzing it in an infrared moisture analyzer. If needed the drying process is then started again.

EXAMPLE 2

Determination of Germination Rate

[0063] To determine the germination rate of a spore powder sample at a point in time the vacuum sealed aluminum bags where the spores were stored at 25° C. were opened and a max. of 1 mg of spores was transferred to an Erlenmeyer flask (150 mL) and mixed into approx. 100 mL water comprising a low percentage of surfactant and agitated with a magnetic stirrer for approx. 30 minutes.

[0064] After that the suspension was diluted 10-100 fold and 100 μL each plated on 2 petri dishes with artificial media (Potatoe dextrose agar+Chloramphenicol+Benomyl). The Petri dishes were incubated for 40 h in darkness at 25° C.

[0065] After that 200 spores were checked for signs of germination (evenly shaped spores, no change of size =no germination, spores showing a germination tube =germinated) and the percentage of germinated spores was calculated.

EXAMPLE 3

Increased Germination Rate of Dried Spores of Metarhizium as Compared to Non-Dried Spores

[0066] Spores of Metarhizium brunneum strain F52 were obtained using solid state fermentation. After separation from the substrate each fermentation batch was divided into fractions each of which was subjected to a vacuum drying procedure to dry to a specific residual moisture content. Spore batches obtained by using two different fermentation substrates, each based on cereals, with subsequent vacuum drying were packaged in sealed aluminum bags and stored until further use. The germination rate was determined according to the procedure described in Example 2.

[0067] As can be seen in FIGS. 1 and 2, Metarhizium spores dried to a residual moisture content of 2 wt.-%, but also of 4 wt.-% showed superior storage stability as compared to spores with higher residual moisture content, in particular a residual moisture content of 12% and above.

EXAMPLE 4

Increased Germination Rate of Dried Spores of Beauveria bassiana as Compared to Non-Dried Spores

[0068] Spores of Beauveria bassiana strain PPRI5339 were obtained using solid state fermentation. After separation from the substrate the spores was divided into fractions each of which was subjected to a vacuum drying procedure to dry to a specific residual moisture content. Spore samples were then stored at 25 or 30° C., respectively and samples taken at certain points in time.

[0069] As can be seen in Table 1 below, the germination rate of the spores did not significantly decrease at a residual moisture content of 4% at 25° C. as compared to a residual moisture content of 8%.

TABLE-US-00001 TABLE 1 Residual Initial Germination Germination moisture Storage germin- rate rate content temperature ation rate (3 months, (6 months, (%) (° C.) (%) %) %) 4 25° C. 87.6% 86.6% 88.8% 4 30° C. 87.6% 80.7% 74.4% 8 25° C. 85.3% 67.9% 54.4% 8 30° C. 85.3% 53.3% 33.4%

Example 5: Increased Germination Rate of Dried Spores of Isaria fumosorosea With Decreased Moisture Content

[0070] Spores of Isaria fumosorosea strain APOPKA97 were obtained using solid state fermentation. After separation from the substrate the spores were divided into fractions each of which was subjected to a vacuum drying procedure to dry to a specific residual moisture content. Spore samples were then stored at 25° C. and samples taken at certain points in time.

[0071] As can be seen in Table 2 below, the germination rate of the spores with the highest moisture level (10%) starts to decrease after 3 months as compared to the other fractions with lower moisture contents.

TABLE-US-00002 TABLE 2 Residual Storage Initial Germination Germination moisture temperature germination rate [1 rate [3 content [%] [° C.] rate [%] month, %] months, %] 2% 25 100 98.77 99.5 3% 25 99.5 99.8 99.51 4% 25 99.5 99.77 97.77 6% 25 99.5 99.04 97.07 10%  25 100 97.31 93.4