METHODS OF INCREASING THE GERMINATION RATE OF FUNGAL SPORES

Abstract

The present invention relates to a method of increasing the germination rate of spores of a fungal microorganisms formulated in a liquid composition essentially free of water, comprising, prior to applying to a plant, plant part or locus where a plant is growing or intended to be grown, suspending said composition in an agriculturally acceptable water-based liquid in a ratio of liquid composition and water-based liquid of between 1000:1 and 1:10. The invention further relates to a method of increasing efficacy of a biological control agent based on spores of a fungal microorganisms and a method of controlling the quality of such biological control agents.

Claims

1. A method of increasing the germination rate of spores of a fungal microorganisms formulated in a liquid composition essentially free of water, comprising, prior to applying to a plant, plant part or locus where a plant is growing or intended to be grown, suspending said composition in an agriculturally acceptable water-based liquid in a ratio of liquid composition:water-based liquid of between 1000:1 and 1:10.

2. A method of increasing efficacy of a biological control agent based on spores of a fungal microorganisms and formulated in a liquid composition essentially free of water, comprising, prior to applying to a plant, plant part or locus where a plant is growing or intended to be grown, suspending said composition in an agriculturally acceptable water-based liquid in a ratio of liquid composition:water-based liquid of between 1000:1 and 1:10.

3. A method of controlling the quality of a biological control agent based on spores of a fungal microorganism formulated in a liquid composition essentially free of water, comprising suspending a sample of said composition in a water-based liquid in a ratio of liquid composition:water-based liquid of between 1000:1 and 1:10 and cultivating a part of the resulting suspension.

4. The method according to claim 1, wherein said spores of a fungal microorganism are conidia.

5. The method according to claim 1, wherein said liquid composition is essentially free of carboxylic acid triglycerides.

6. The method according to claim 1, wherein said liquid composition comprises at least one surfactant.

7. The method according to claim 6, wherein said liquid composition comprises at least 1%, preferably at least 5 wt.-% of said at least one surfactant.

8. The method according to claim 7, wherein said liquid composition comprises at least 50 wt.-% of said at least one surfactant.

9. The method according to claim 3, wherein said water further comprises an agent which decreases or prevents hyphal growth.

10. The method according to claim 9, wherein said agent is benomyl.

11. The method according to claim 1, wherein said suspension is to be kept for at least 10 seconds, preferably at least 1 minute prior to application or cultivation.

12. The method according to claim 3, wherein the ratio of composition and water is between 50:1 and 1:5.

13. The method according to claim 12, further comprising, after said suspending, further diluting the suspension.

14. The method according claim 6, wherein said surfactant is a polyether-modified trisiloxane.

15. The method according to claim 14, wherein said surfactant is BREAKTHRU® S240 or BREAKTHRU® S245.

16. The method according claim 1, wherein said fungal microorganism is useful in agriculture.

17. The method according to claim 1, wherein the germination rate of said fungal microorganism is increased by at least 5% as compared to the germination rate of the same fungal microorganism comprised in a liquid composition suspended in a water-based liquid in a ratio of 1:99.

18. The method according to claim 1, wherein said fungal microorganism is Purpureocillium lilacinum.

19. The method according to claim 1, wherein said liquid composition and/or the water based liquid have a temperature of at least 12° C.

20. The method according to claim 1, wherein said liquid composition is essentially free of mineral oil.

21. The method according to claim 1, wherein said liquid composition is essentially free of oil.

Description

EXAMPLE 1

Materials and Methods

Production of Spores and Spore Formulations

[0108] Fermentation of P. lilacinum strain 251 and I. fumosorosea strain Apopka 97 was carried out in a modular solid state fermenter as described in US6620614. Harvested spores were dried to a residual moisture content of <10%. P. lilacinum strain 251 spores were dispersed in BreakThru S245 (final formulation comprising about 74 wt.-% BreakThruS240 and about 6% Aerosil) yielding formulations containing >5.5E+10 spores/ml. I. fumosorosea strain Apopka 97 spores were dispersed in Tween 20 (final formulation comprising about 95 wt.-% Tween 20, about 2.5wt.-% Aerosil 200 and about 2.5% spores) yielding formulations containing >5.0E+09 spores/ml.

Determination of the Germination Rate

[0109] To determine the germination rate of fungal spores, water-based spore suspensions were generated. If not otherwise indicated, for the control samples, the liquid composition (formulated spores) was suspended with pure water in a ratio of composition and water of 1:99. Both the water and the composition were equilibrated to 22° C. before mixing. Also the next steps were conducted at 22° C. The suspensions were stirred with magnetic stir bars for at least 15 minutes (700 rpm) before spore suspensions were further diluted in water and spread on PDA (potato dextrose agar) plates or PDA plates containing 20 μg/ml benomyl. The plates were incubated at 25° C. for 1 to 2 days until germination was monitored microscopically. The fungistatic compound benomyl which does not inhibit spore germination but inhibits hyphal elongation allows tracking spore germination after prolonged incubation times because the not germinated spores are not overgrown by hyphae of the germinated spores. To test the influence of the ratio of liquid composition and water on the ability of the spores to germinate, the liquid composition was suspended with varying volumes of pure water (see examples below). If not otherwise indicated, both the water and the composition were equilibrated to 22° C. before mixing. Such a suspension, hereinafter also called “imbibition premix” was then further diluted with 22° C. temperated water to reach the same ratio of composition and water as in the control samples, i.e 1:99. The subsequent processing of samples derived from an imbibition premix were the same as those for the controls.

EXAMPLE 2

The Ratio of Liquid Composition and Water During Imbibition Impacts the Germination Rate of Formulated Spores

[0110] Spores of P. lilacinum strain 251 were produced, dried, and formulated in BreakThru S245 as described in Example 1. Eventually, batches of formulated spores were stored for prolonged time at 30° C. as indicated in Table 1. From the formulated spore batches several imbibition premixes were created ranging from a ratio of liquid composition and water from 1000:1 to 1:10. The premixes were held for two hours at 22° C. before they were diluted to the same ratio of liquid composition and water as in the control (see Example 1). The ability of the spores to germinate was then monitored after 1 day incubation on PDA as described in Example 1. Compared to the control, in which the spore suspension was created with a ratio of liquid composition and water of 1:99, imbibition premixes of bigger ratios could improve the germination rate by more than 90% (Table 1). Ratios of liquid composition and water between 100:1 and 1:5 yielded particularly improved germination rates.

TABLE-US-00001 TABLE 1 The ratio of liquid composition and water during imbibition impacts the germination rate of formulated spores. formulated spores formulated spores formulated spores formulated spores batch #1 batch #2 batch #3 batch #4 8 months @ 30° C. 8 months @ 30° C. 10 months @ 30° C. 0 months storage ratio liquid Germination.sup.a STD Germination.sup.a STD Germination.sup.a STD Germination.sup.a STD composition:water (%) Ev.sup.b (%) EV.sup.b (%) EV.sup.b (%) EV.sup.b 1000:1   NA NA NA NA 64.4 0.8 NA NA 333:1  NA NA NA NA 67.4 0.1 NA NA 100:1  63.8 7.6 54.9 1.6 74.0 1.8 88.6 0.1 33:1  85.6 1.1 82.0 1.9 79.5 1.3 94.4 1.0 10:1  91.5 0.0 87.5 2.5 80.2 0.2 97.6 0.7 1:1 79.3 0.9 79.9 1.1 77.0 0.1 92.5 0.1 1:5 78.8 0.5 68.8 1.1 NA NA 83.8 1.0  1:10 69.6 1.7 48.3 0.3 NA NA 66.7 3.2 control 47.8 2.9 57.4 3.7 51.1 2.6 82.4 0.8 .sup.aAverage of two replicates; .sup.bStandard deviation of two replicates; the notation “NA” denotes that the data point was not determined

EXAMPLE 3

Impact of Imbibition Duration on the Germination Rate of Formulated Spores

[0111] Spores of P. lilacinum strain 251 were produced, dried, and formulated in BreakThru S245 as described in Example 1. Eventually, batches of formulated spores were stored for prolonged time at 30° C. as indicated in Table 2. Imbibition premixes were prepared as described in Example 1, and the premixes were held for various durations at 22° C. as shown in Table 2. The ability of the spores to germinate was then monitored after 1 day incubation on PDA as described in Example 1. The results of this example demonstrate that short imbibitions of 1 minute in an imbibition premix are sufficient to increase the germination rate by more than 80% compared to control imbibitions (Table 2).

TABLE-US-00002 TABLE 2 Impact of imbibition duration at various liquid composition: water ratios on the germination rate of formulated spores. imbibition formulated spores #3 ratio liquid premix 10 months @ 30° C. composition: holding time Germination.sup.a water (min) (%) STDEV.sup.b 10:1  1 52.9 0.5  5 65.8 1.6 30 63.3 1.0  1:1  1 65.9 1.8  5 64.6 1.1 30 48.6 0.6 Control 36.0 0.8 .sup.aAverage of two replicates; .sup.bStandard deviation of two replicates

EXAMPLE 4

Impact of Imbibition Temperature on the Germination Rate of Formulated Spores

[0112] Spores of P. lilacinum strain 251 were produced, dried, and formulated in BreakThru S245 as described in Example 1. Eventually, batches of formulated spores were stored for prolonged time at 30° C. as indicated in Table 3. Imbibition premixes were prepared essentially as described in Example 1, but in this example, sub samples of a batch of formulated spores were equilibrated to the temperatures 4° C., 12° C., 22° C. and 37°. The water used for the respective imbibition was equilibrated to the same temperatures, and only equally temperated liquid compositions and water were mixed. Thus, the initial temperature for preparing the imbibition premix corresponded to 4° C., 12° C., 22° C. and 37° C., as indicated in Table 3. However since the mixing and the subsequent handling of the samples occurred at an surrounding temperature of 22° C. the temperature of the samples most likely changed towards 22° C. over time. This assumed temperature shift was not monitored. All premixes were hold for 30 min before the ability of the spores to germinate was monitored after 1 day incubation on PDA as described in Example 1. The results showed that warmer imbibitions increased the germination rate compared to colder imbibitions (Table 3). Compared to control imbibitions, which were conducted at 22° C., an imbibition premix at 12° C. already yielded an increased germination rate by 36%; the germination rate could be even further increased by preparing the imbibition premix at temperatures of 22° C. and above (Table 3).

TABLE-US-00003 TABLE 3 Impact of imbibition temperature of various liquid composition: water ratios on the germination rate of formulated spores. formulated spores initial temperature 10 months @ 30° C. ratio liquid of imbibition Germination.sup.a composition:water premix (%) STDEV.sup.b 10:1  4° C. 53.4 0.7 12° C. 69.9 1.7 22° C. 72.6 4.7 37° C. 77.2 2.6  1:1  4° C. 56.3 0.4 12° C. 70.5 1.8 22° C. 76.1 2.8 37° C. 77.8 1.1 control 51.1 2.6 .sup.aAverage of two replicates; .sup.bStandard deviation of two replicates

EXAMPLE 5

Spore Content in Liquid Composition Hardly Influences the Germination Rate

[0113] Spores of P. lilacinum strain 251 were produced, dried, and formulated in BreakThru S245 as described in Example 1. Eventually, batches of formulated spores were stored for prolonged time at 30° C. as indicated in Table 4. Sub samples of a batch of formulated spores were further diluted with BreakThru S245 to lower the fraction of spores in the formulation from 20% to 10% and 2%, after which imbibition premixes were prepared as shown in Table 4 and described in Example 1. As imbibition control served the original composition with 20% spore fraction. All imbibitions were conducted at 22° C. and the premixes were held for 30 min before the ability of the spores to germinate was monitored after 1 day incubation on PDA as described in Example 1. The results did not reveal clear hints that the amount of spores in the formulation determines the ability of the imbibition premix to increase the germination rate: compared to the control, the premixes with a ratio of liquid composition and water of 1:1 increased the germination by 79%-100% and the premixes with a ratio of liquid composition and water of 10:1 increased the germination by 109%-123% (Table 4). From this data, it can be concluded that rather the ratio of liquid composition and water is decisive for the increased germination and not the ratio of spores and water.

TABLE-US-00004 TABLE 4 Impact of the spore fraction in formulation on the ability to germinate. fraction of formulated spores spores in ratio liquid 9 months @ 30° C. formulation composition: Germination.sup.a (%) water (%) STDEV.sup.b 20% 10:1 76.9 1.1 10% 81.8 3.1  2% 76.8 0.0 20%  1:1 65.8 0.4 10% 73.4 0.8  2% 68.3 3.1 control 36.7 3.2 .sup.aAverage of two replicates; .sup.bStandard deviation of two replicates

EXAMPLE 6

The Ratio of Liquid Composition and Water During Imbibition Impacts the Germination Rate of I. Fumosorosea Spores Formulated in the Surfactant Tween 20

[0114] Spores of I. fumosorosea strain Apopka 97 were produced, dried, and formulated in the carrier liquid Tween 20 as described in Example 1. The formulated spores were stored in closed bottles at 40° C. for 2 months before subjecting them to quality control. Imbibition premixes were prepared as described in Example 1, and the premixes were held for 30 minutes at 22° C. before further dilution. The ability of the spores to germinate was then monitored after 1 day incubation on PDA. The results of this example demonstrate that imbibition premixes of ratios of liquid composition and water of 10:1 and 1:1 lead to improved germination rates compared to spore suspensions prepared in the control ratio of 1:99 (Table 5).

TABLE-US-00005 TABLE 5 The ratio of liquid composition and water during imbibition impacts the germination rate of I. fumosorosea spores formulated in Tween 20 after storage. formulated spores 6 months storage ratio liquid at 30° C. composition: Germination.sup.a water (%) STDEV.sup.b 10:1 89.3 2.0  1:1 91.2 0.4 control 86.1 0.1 .sup.aAverage of two replicates; .sup.bStandard deviation of two replicates; the notation “NA” denotes that the data point was not determined

EXAMPLE 7

The Ratio of Liquid Composition and Water During Imbibition Impacts the Germination Rate of M. Brunneum Spores Formulated in Petroleum Distillates

[0115] A commercial product containing >2E+09 spores/g of M. brunneum strain F52 formulated in a petroleum based liquid was stored for prolonged time of 12 months at 20° C. after which the bottle has been opened and closed under normal atmosphere, followed by storage of another 14 months at 4° C. Thus, total storage time was 26 months before subjecting them to quality control. Imbibition premixes were prepared as described in Example 1, and the premixes were held for 30 minutes at 22° C. before further dilution. The ability of the spores to germinate was then monitored after 1 day incubation on PDA. Compared to control imbibitions at a ratio of liquid composition and water of 1:99, imbibition premixes of ratios of 1:1 and in particular 10:1 improved germination rates (Table 6).

TABLE-US-00006 TABLE 6 The ratio of liquid composition and water during imbibition impacts the germination rate of M. brunneum spores formulated in petroleum distillates after prolonged storage. formulated spores ratio liquid 26 months storage composition: Germination.sup.a water (%) STDEV.sup.b 10:1 37.2 3.0  1:1 27.3 2.6 control  9.8 0.2 .sup.aAverage of two replicates; .sup.bStandard deviation of two replicates; the notation “NA” denotes that the data point was not determined