METHOD OF IMPROVING STORAGE STABILITY AND FITNESS OF FUNGAL SPORES

Abstract

The present invention relates to a method for producing dormant fungal structures or organs with an improved germination rate comprising subjecting said dormant structures or organs to a procedure comprising a heat treatment, followed by a cooling period as well as a related solid-state fermentation method and dormant fungal structures or organs produced thereby.

Claims

1. A method for producing dormant fungal structures or organs with an improved germination rate comprising subjecting said dormant structures or organs to a procedure comprising a heat treatment of between 37 C. and 65 C., followed by a cooling period to a temperature of between 0 C. and 36 C.

2. The method according to claim 1, further comprising producing said dormant fungal structures or organs by fermentation.

3. The method according to claim 2, wherein said dormant fungal structures or organs are subjected to said procedure during or after fermentation.

4. The method according to claim 1, wherein said dormant fungal structure or organs are exospores or spores which are developing spores or mature spores.

5. (canceled)

6. The method according to claim 2, wherein said fermentation is solid-state fermentation.

7. The method according to claim 1, wherein said dormant fungal structures or organs are spores of at least one filamentous fungus.

8. The method according to claim 7, wherein said at least one filamentous fungus is an entomopathogenic fungus.

9. The method according to claim 8, wherein said entomopathogenic fungus is of the genus Metarhizium.

10. The method according to claim 9, wherein said entomopathogenic fungus is of the species Metarhizium brunneum and/or Metarhizium acridum.

11. The method according to claim 8, wherein said entomopathogenic fungus is selected from the group consisting of Beauveria bassiana; Lecanicillium lecanii; Lecanicillium muscarium; Metarhizium brunneum; M. acridum ARSEF324; M. acridum isolate IMI 330189 (ARSEF7486); Nomuraea rileyi; Isaria fumosorosea strain apopka 97, Isaria fumosorosea strain FE 9901; and Beauveria brongniartii.

12. The method according to claim 7, wherein said at least one filamentous fungus is selected from the group consisting of a plant growth promoting fungus, a fungus active against plant pathogens, a fungus active against nematodes and a fungus having herbicidal activity.

13. The method according to claim 12, wherein said plant growth promoting fungus is selected from the group consisting of Talaromyces flavus, Trichoderma atroviride; Trichoderma harzianum; Penicillium bilaii; Pythium oligandrum; Rhizopogon amylopogon; Rhizopogon fulvigleba; Trichoderma harzianum; Trichoderma koningii; Trichoderma virens; and Verticillium albo-atrum.

14. (canceled)

15. The method according to claim 1, wherein said heat treatment comprises an elevation of the temperature within a spore containing vessel to between 37 and 55 C.

16. The method according to claim 1, wherein said dormant structure or organs are fungal spores.

17. The method according to claim 1, wherein said heat treatment is effected for at least 30 minutes.

18. The method according to claim 1, wherein said cooling period is at between 5 C. and 36 C.

19. The method according to claim 1, wherein said cooling period lasts at least 6 hours.

20. The method according to claim 1, wherein said dormant fungal structures or organs exhibit an increased germination rate as compared to dormant fungal structures or organs not subjected to said procedure after 2 weeks.

21. A composition comprising dormant fungal structures or organs having an improved germination rate, said composition comprising a) a carrier; and b) dormant fungal structures or organs subjected to a procedure comprising a heat treatment at a temperature of between 37 C. and 65 C. followed by a cooling period at a temperature of between 0 C. and 36 C., wherein 2 weeks after finishing said procedure said dormant fungal structures or organs exhibit an improved germination rate and/or germination efficiency as compared to dormant fungal structures or organs not subjected to the treatment in step b).

22. A storage-stable composition comprising dormant fungal structures or organs that are fungal spores which have been produced according to the method of claim 1.

23-26. (canceled)

Description

EXAMPLE 1: MATERIALS AND METHODS

Determination of the Germination Rate

[0061] To determine the germination rate of fungal spores, water-based spore suspensions were generated. For example, conidia were harvested from agar plates by flooding the plate with water supplemented with 0.1% of the detergent Neo-wett (Kwizda Agro) and the spores scraped off using cell scrapers. These suspensions were passed through 50 m strainers to remove fungal hyphae. Alternatively, spores were produced by solid state fermentation and harvested as described below. All harvested spores were spread on PDA (potato dextrose agar) plates and incubated at 25 C. for 1 to 2 days until germination was monitored microscopically.

Determination of Metabolic Activity

[0062] The metabolic activity of fungal spores in a nutrient containing environment was determined using the Presto Blue Cell Viability reagent (Invitrogen). This resazurin-based assay can monitor in a linear fashion the metabolic activity of cell populations (Hamalainen-Laanaya and Orloff, 2012. Analysis of cell viability using time-dependent increase in fluorescence intensity. Analytical Biochemistry 429 (1), pp: 32-38). To measure the activity of fungal spores, spore suspensions were generated as described above, and serial 1:2 dilutions of spores were subjected to PDB (potato dextrose broth) containing 10% Presto Blue Cell Viability reagent. This suspension was incubated 16 to 48 hours at 25 C. before fluorescence was measured according to the manufactures recommendation. The serial dilutions were included to make sure that the metabolic activity was monitored in the linear range of the Presto Blue Cell Viability assay. The arbitrary units of the fluorescence measurements were normalized to the number of spores in each sample. Spores were counted by cell counting methods well known in the art such as haemocytometer.

Determination of the Temperature Tolerance

[0063] To determine the tolerance of fungal spores to elevated temperatures, spore suspensions were incubated at 44 C. for 0 min (control), 5 min, 10 min, 15 min, 20 min, 30 min and 60 min before subjecting them to the Presto Blue assay as described above. Curves were fit with the Boltzmann sigmoid equation to determine the time of 50% inhibition (IT50).

Solid-State Fermentation of Fungal Spores on the Example of Metarhizium brunneum F52

[0064] Fermentation of the M. brunneum strain F52 was performed in a modular solid state fermenter according to Liith and Eiben (see U.S. Pat. No. 6,620,614) with 1.5 kg grain-based cultivation substrate per module base and a constant airflow. The fermenter was placed in a room with a temperature of 22 C. The cooling system of the fermenter, which consisted of a cooling coil within each module base according to Liith and Eiben, was adjusted in such way that the cooling liquid was pumped through the cooling coil, when 25 C. were exceeded in the cultivation substrate, until it had cooled down again to 20 C. To apply the heat treatment during fermentation, the cooling liquid was replaced with 41 C. hot liquid, which resulted in a maximum temperature of 40 C. in the cultivation substrate. After termination of fermentation the spores were harvested by vacuuming and cyclonic separation. The spore powder was sieved (40 m pore size) to remove fungal hyphae and residues of the cultivation substrate. Further processing of the harvested conidia included vacuum-drying which increased the relative dry mass of the spores from about 50% to about 92%. This drying step was applied in studies monitoring the long term shelf life of fungal spores for 3 months and longer.

EXAMPLE 2: INFLUENCE OF A HEAT TREATMENT AT DIFFERENT TEMPERATURES ON CONIDIA ON THE EXAMPLES OF M. BRUNNEUM

Protocol:

[0065] Spores of the M. brunneum strain F52 were spread on a PDA plate, and plates were incubated at 25 C. to allow germination, mycelial growth and formation of a new generation of conida, respectively. 12 days after inoculation, a timepoint at which conidiogenesis was completed, the plates were shifted to higher temperatures, i.e. 35 C., 37 C., 38 C., 39 C., 40 C., 41 C., 42 C. and 43 C. for 12 h. After this treatment plates were placed back to 25 C. Control plates were constantly incubated at 25 C. 14 days after inoculation (dai), conidia were harvested from the plates as described in Material & Methods. For determining the metabolic activity and the temperature tolerance of the respective spore suspensions, the Presto Blue assay was utilized (see Material & Methods). To study the storage stability of the spores, the suspensions were centrifuged and the supernatant was discarded. The spores were air-dried for several hours at room temperature, and then subjected to 30 C. for 1 week. The germination rate before and after this storage period was determined by incubating the spores for 20 h on PDA plates at 25 C. as described in Material & Methods. All assays were performed with two biological replicates.

Results:

[0066] The investigation of the temperature tolerance and the short term storage stability (Table 1) revealed that applying a heat treatment to the spores can enhance the temperature tolerance of the spores as well as their storage stability. With respect to temperature tolerance we observed a more than three-fold increase of the IT50 at 44 C. (Table 1), and with respect to the storage stability we observed an about 10 fold increased germination rate, i.e. an increase from 1.9% germination of control spores to up to 18% germination after 1 week storage at 30 C. (Table 1). This effect showed a clear temperature dependency with regard to the heat treatment. While a treatment at 35 C. did hardly show an effect, the effect emerged at 37 C. and was most pronounced between 38 C. and 41 C. For this heat treatment setup of M. brunneum F52, temperatures above 41 C. led to an inhibition of the metabolic activity and germination.

TABLE-US-00001 TABLE 1 Influence of a heat treatment at different temperatures on conidia on the examples of M. brunneum. Germination after 1 week Metabolic IT50 at Germ. storage @ activity 44 C. sample (%) STDEV 30 C. (%) STDEV (a.u.) STDEV (min) STDEV ctrl 97.4 0.1 1.9 0.9 9.9 0.3 11.3 0.6 35 C. 99.0 0.3 2.9 1.2 9.5 0.5 20.2 1.0 37 C. 99.8 0.3 7.2 2.3 10.0 0.5 27.3 0.6 38 C. 99.1 1.3 10.5 5.7 9.9 1.1 33.9 0.5 39 C. 99.3 1.0 18.1 5.7 9.4 2.0 34.3 0.9 40 C. 99.2 1.1 15.2 1.7 8.7 0.0 34.9 0.3 41 C. 99.0 0.0 14.1 4.3 9.2 0.1 30.1 0.3 42 C. 86.7 2.3 7.6 3.1 7.4 0.1 28.3 3.5 43 C. 40.6 28.8 1.1 0.9 2.6 2.4 22.8 5.4 Germ.: Germination; STDEV: standard deviation; a.u.: arbitrary units

EXAMPLE 3: INFLUENCE OF THE DURATION OF A HEAT TREATMENT ON CONIDIA ON THE EXAMPLES OF M. BRUNNEUM

Protocol:

[0067] Spores of the M. brunneum strain F52 were spread on a PDA plate, and plates were incubated at 25 C. to allow germination, mycelial growth and formation of a new generation of conida, respectively. 12 days after inoculation, a timepoint at which conidiogenesis was completed, the plates were shifted to a temperature of 40 C. for 1 h, 3 h, 6 h 12 h, and 24 h, respectively. After this treatment plates were placed back to 25 C. Control plates were constantly incubated at 25 C. 14 days after inoculation, conidia were harvested and the metabolic activity and the temperature tolerance were determined as described in Material & Methods. To study the storage stability of the spores, the suspensions were centrifuged and the supernatant was discarded. The spores were dried at the air for 2 to 4 hours at room temperature, and then subjected to 30 C. for 1 week. The germination rate before and after this storage period was determined by incubating the spores for 20 h on PDA plates at 25 C. as described in Material & Methods. All assays were performed with two biological replicates.

Results:

[0068] The investigation of the temperature tolerance and the short term storage stability (Table 2) revealed that a 40 C. treatment of 1 h was sufficient to increase temperature tolerance as well as storage stability. However these effects were most pronounced when the 40 C. treatment was applied for a time between 3 h and 12 h (Table 2), in such cases the temperature tolerance, i.e. the IT50 at 44 C., was increased almost three-fold, and the germination rate after storage at 30 C. for one week increased about five-fold, i.e. from about 1% to about 5%.

TABLE-US-00002 TABLE 2 Influence of the duration of a heat treatment on conidia on the examples of M. brunneum Germination after 2 weeks Metabolic IT50 at Germ. storage @ activity 44 C. sample (%) STDEV 30 C. (%) STDEV (a.u.) STDEV (min) STDEV ctrl 98.1 0.0 1.0 0.0 14.3 1.2 8.3 0.3 1 h 96.8 1.7 1.7 0.3 14.1 0.4 13.7 1.1 3 h 99.3 0.4 4.0 0.4 14.5 0.1 20.4 2.1 6 h 94.4 0.1 4.9 0.3 13.7 0.4 21.7 0.4 12 h 94.2 2.5 3.1 1.0 13.4 0.6 18.4 1.3 24 h 70.8 0.5 1.0 0.0 8.0 1.4 12.5 0.5

EXAMPLE 4: INFLUENCE OF THE TIMEPOINT OF HEAT TREATMENT ON CONIDIA ON THE EXAMPLES OF M. BRUNNEUM

Protocol:

[0069] Spores of the M. brunneum strain F52 were spread on a PDA plate, and plates were incubated at 25 C. Plates were subjected to a heat treatment at 40 C. for 6 h after which temperature was lowered to 25 C. again. These temperature shifts were performed at different days after inoculation, i.e. on day 7, 9, 11, 12 and 13. Control plates were constantly incubated at 25 C. 14 days after inoculation, conidia were harvested and the metabolic activity and the temperature tolerance were determined as described in Material & Methods. To study the storage stability of the spores, the suspensions were centrifuged and the supernatant was discarded. The spores were air-dried for 2 to 4 hours at room temperature, and then subjected to 30 C. for 1 week. The germination rate before and after this storage period was determined by incubating the spores for 20 h on PDA plates at 25 C. as described in Material & Methods. All assays were performed with two biological replicates.

Results:

[0070] The investigation of the temperature tolerance and the short term storage stability (Table 3) revealed that any of the time points during fungal development at which the 40 C. treatment was applied resulted in spores which were more tolerant towards elevated temperature, i.e. the IT50 at 44 C. increased about three-fold, and had increased storage stability, i.e. the germination rate after storage at 30 C. for one week increased from less than 1% to 5-8% depending on the time of application of the heat treatment. The highest increase of the storage stability was observed when the heat treatment was applied just one day before harvest (Table 3). Notably, applying the heat treatment at early time points, such as 5 and 7 days before harvest, the spore yield was negatively affected (Table 3), likely reflecting a dysfunction during conidiogenesis.

TABLE-US-00003 TABLE 3 Influence of the timepoint of heat treatment on conidia on the examples of M. brunneum. Germination after 1 week Metabolic IT50 at Yield Germ. storage @ activity 44 C. (spores sample (%) STDEV 30 C. (%) STDEV (a.u.) STDEV (min) STDEV per plate) STDEV ctrl 98.3 0.4 0.2 0.3 16.4 1.3 14.6 0.8 4.02E+09 3.42E+08 7 dai 97.8 0.3 5.0 0.3 15.9 0.7 33.6 3.2 2.56E+09 1.43E+08 9 dai 98.3 0.2 5.1 0.3 16.7 0.2 36.6 3.5 3.05E+09 1.12E+09 11 dai 98.7 0.3 7.0 0.6 17.2 1.4 36.6 1.2 3.79E+09 3.46E+07 12 dai 98.3 0.5 6.4 1.2 18.4 0.2 37.7 0.6 3.76E+09 6.87E+08 13 dai 98.4 0.7 8.1 0.3 16.5 0.4 40.6 3.0 4.14E+09 4.10E+08 dai: days after inoculation.

EXAMPLE 5: INFLUENCE OF A RECOVERY PHASE AFTER HEAT TREATMENT ON CONIDIA ON THE EXAMPLES OF M. BRUNNEUM (2)

Protocol:

[0071] Spores of the M. brunneum strain F52 were spread on PDA plates, and plates were incubated at 25 C. Spores were shifted to 40 C. for 12 h on day 13 after inoculation, i.e. 24 h before harvest. These spores were compared with spores which were shifted to 40 C. for 12 h on day 14 after inoculation, i.e. the heat treatment has been performed directly before harvest. The harvested conidia were subjected to the Presto Blue metabolic activity assay as described in Material & Methods. To study the storage stability of the spores, the suspensions were centrifuged and the supernatant was discarded. The spores were air-dried for 2 to 4 hours at room temperature, and then subjected to 30 C. for 2 weeks. The germination rate before and after this storage period was determined by incubating the spores for 20 h on PDA plates at 25 C. as described in Material & Methods. All assays were performed with two biological replicates.

Results:

[0072] The investigation of the metabolic activity and the short term storage stability (Table 4) revealed that the spores which were heat treated directly before harvest were strongly affected in their metabolic activity and also showed reduced germination (Tables 4). Thus, a recovery phase is necessary to build the desired traits of spore robustness after heat treatment.

TABLE-US-00004 TABLE 4 Influence of a recovery phase after heat treatment on conidia on the examples of M. brunneum Germination Germi- after 1 week Metabolic nation storage @ activity (%) STDEV 30 C. (%) STDEV (a.u.) STDEV dai 13 97.3 0.4 17.8 2.9 11.3 0.5 dai 14 43.9 3.7 0.5 0.7 1.1 0.5 (no re- covery)

EXAMPLE 6: INFLUENCE OF RECOVERY TEMPERATURE AND DURATION AFTER HEAT TREATMENT ON CONIDIA ON THE EXAMPLE OF M. BRUNNEUM (2)

Protocol:

[0073] Spores of the M. brunneum strain F52 were spread on a PDA plate, and plates were incubated at 25 C. Either 10 days after inoculation or 12 days after inoculation, plates were subjected to a heat treatment at 40 C. for 6 h after which temperature was lowered to either 25 C. or 10 C. Control plates were constantly incubated at 25 C. 14 days after inoculation, conidia were harvested and the metabolic activity and the temperature tolerance were determined as described in Material & Methods. To study the storage stability of the spores, the suspensions were centrifuged and the supernatant was discarded. The spores were air-dried for 2 to 4 hours at room temperature, and then subjected to 30 C. for 1 week. The germination rate before and after this storage period was determined by incubating the spores for 20 h on PDA plates at 25 C. as described in Material & Methods.

Results:

[0074] The analysis revealed that a recovery phase at 10 C. for 2 days was not sufficient, reflected by the inability to germinate after 1 week storage at 30 C. (Tables 5). By contrast, increasing the recovery phase at 10 C. from 2 days to 4 days, a strong increase in storage stability was observed when compared to the control spores. This increase was even more pronounced than the increased storage stability using a recovery temperature of 25 C. (Table 5).

TABLE-US-00005 TABLE 5 Influence of a recovery temperature after heat treatment on conidia on the examples of M. brunneum Germination after 1 week Germination storage @ (%) STDEV.sup.a 30 C. (%) STDEV.sup.a ctrl 98.8 #N/V 5.9 #N/V 2 days 25 C. 98.3 #N/V 13.8 #N/V recovery 4 days 25 C. 96.9 #N/V 14.0 #N/V recovery 2 days 10 C. 89.3 #N/V 0.0 #N/V recovery 4 days 10 C. 95.5 #N/V 17.3 #N/V recovery .sup.athe assay was not performed in replicates

EXAMPLE 7: EFFECT OF A HEAT TREATMENT IN LARGE-SCALE FERMENTATION ON THE EXAMPLE OF M. BRUNNEUM

Protocol:

[0075] Two different batches of spores of the M. brunneum strain F52 were produced by solid state fermentation as described in Example 1. In one batch, the fermenter was heated for 12 h with 41 C. hot liquid at day 19 after inoculation. After this heat treatment, the fermenter was allowed to cool down to room temperature (about 22 C.). 21 days after inoculation the conida were harvested. Thus, the recovery phase constituted almost 2 days. The other batch was a conventional fermentation run without any heat treatment. Details on the fermentation and harvest procedures are given in the Material & Methods section. Aliquots of vacuum-dried spores were vacuum-sealed in aluminum bags and stored at 25 C. The spores stored in this way were subjected to germination assays at different timepoints. For selected samples the metabolic activity was determined using the Presto Blue assay.

Results:

[0076] The results revealed an improved germination rate through the heat treatment of the fermenter (Table 6), i.e. germination was 1.2-fold increased after 2 weeks storage, two-fold increased after 3 months, and six-fold increased after 6 months. Furthermore, measuring the metabolic activity of the spores after 6 months storage at 25 C. revealed an 18-fold higher metabolic activity of the spores derived from the heat treated fermenter (Table 6).

TABLE-US-00006 TABLE 6 Effect of a heat treatment in large-scale fermentation on the example of M. brunneum Metabolic Germination after storage at 25 C. (%) activity (a.u.) 0 weeks.sup.a 2 weeks.sup.a 3 months.sup.b 6 months.sup.b 6 months F52 92.0 65.4 33.1 8.5 4.5 F52 + 92.3 79.4 65.4 54.3 82.7 heat .sup.aGermination of spores was determined after 20 h incubation on PDA at 25 C. .sup.bGermination of spores was determined after 40 h incubation on PDA at 25 C.

EXAMPLE 8: EFFECT OF A HEAT TREATMENT ON A DIFFERENT FUNGAL STRAIN

Protocol:

[0077] Spores of the Metarhizium acridum strain ARSEF324 (the active ingredient of Green Guard) were spread on PDA plates, and plates were incubated at 25 C. 12 days after inoculation, a time point at which conidiogenesis was completed, the plates were shifted to 40 C. for 6 h. After this treatment plates were placed back to 25 C. Control plates were constantly incubated at 25 C. 14 days after inoculation, conidia were harvested from the plates as described in Material & Methods. For determining the metabolic activity and the temperature tolerance of the respective spore suspensions, the Presto Blue assay was utilized (see Material & Methods). Since the ARSEF324 strain is described to be generally more temperature tolerant than the F52 strain, a slightly different setup was chosen: the spore suspensions were incubated at 44 C. for 0 min (control), 5 min, 10 min, 20 min, 30 min, 60 min and 120 min. To study the storage stability of the spores, suspensions were centrifuged and the supernatant was discarded. The spores were air-dried for 2 to 4 hours at room temperature, and then subjected to 30 C. for 2 weeks. The germination rate before and after this storage period was determined by incubating the spores for 20 h on PDA plates at 25 C. as described in Material & Methods. All assays were performed with two biological replicates.

Results:

[0078] The investigation of the temperature tolerance and the short term storage stability (Table 7) revealed that both traits were improved through the heat treatment (Table 7). Additionally, it was observed that the metabolic activity of the heat treated spores was more than two-fold increased, indicating that the heat treatment in this isolate generally stimulates vitality.

TABLE-US-00007 TABLE 7 Effect of a heat treatment on a different fungal strain Germination after 2 weeks Metabolic IT50 at Germination storage @ activity 44 C. sample (%) 30 C. (%) STDEV (a.u.) STDEV (min) STDEV ARSEF 97.6 59.6 11.2 7.7 0.7 30.6 1.2 324 ARSEF 97.2 87.4 5.15 20.3 6.9 62.6 13 324 + heat