WO2005019436A1

WO2005019436A1 - Trichoderma composition

Info

Publication number: WO2005019436A1
Application number: PCT/IB2003/003531
Authority: WO
Inventors: Siska Janse Van Vuuren; Regardt Freeswijk
Original assignee: Axiom Bio Products (Proprietary) Limited
Priority date: 2001-11-09
Filing date: 2003-08-26
Publication date: 2005-03-03
Also published as: ZA200301049B

Abstract

A method of producing a liquid Trichoderma composition includes the steps of growing Trichoderma on a growth medium, transferring the Trichoderma from the growth medium to an aqueous growth medium which contains a first Trichoderma food source, allowing Trichoderma growth to continue and then diluting the aqueous growth medium with a diluent which contains a second Trichoderma food source. The first Trichoderma food source is selected from malt extract, peptone and mixtures thereof and the second Trichoderma food source is selected from molasses, yeast, malt extract and mixtures of any two or more thereof.

Description

TRICHODERMA COMPOSITION

THIS INVENTION relates to a method of producing a liquid Trichoderma composition and to a liquid Trichoderma composition. It also relates to a method of treating a plant disease.

Trichoderma is a naturally occurring fungus which is found in soil. The use of Trichoderma for the control of plant diseases and other pathogenic fungi is well known. Trichoderma is also used to promote general plant growth. Trichoderma is commonly produced by growing the fungus in a liquid medium until a viable density has been reached and then harvesting and drying the Trichoderma spores. The spores are , generally air-dried and the product is a powder containing spores of Trichoderma. For the purpose of this specification, the word "spores" should be understood to include within its scope conidia or chlamydospores or both. Conidia are asexual fungal spores produced exogenously from a hyphal tip. Chlamydospores are hyphal cells that become thick-walled and separate from the parent mycelium and function as spores.

Prior art methods have not produced stable liquid compositions containing live Trichoderma spores. It is an object of this invention to provide a stable liquid composition containing live conidia and/or chlamydospores of Trichoderma species. Prior art formulations have been unable to keep Trichoderma spores alive. In particular, it is an object of this invention to provide a liquid formulation, in which Trichoderma spores remain alive for an extended period of 6 - 12 months or longer.

According to a first aspect of the invention there is provided a method of producing a liquid Trichoderma composition, the method including the steps of growing Trichoderma on a growth medium; transferring the Trichoderma from the growth medium to an aqueous growth medium which contains a first Trichoderma food source and allowing Trichoderma growth to continue; and diluting the aqueous growth medium with a diluent which contains a second Trichoderma food source. .

The growth medium may be a gel growth medium such as an Agar medium.

The first Trichoderma food source may be selected from malt extract (e.g. bacteriological malt extract), peptone (e.g. bacteriological peptone) and mixtures thereof.

Typically, an inoculant of a desired Trichoderma strain is prepared and grown on a gel medium containing agar for between one and four days either on a flat surface or in a test-tube. The Trichoderma are then harvested and placed in the liquid growth medium. Optionally, the mycelia and conidia are transferred to a smaller scale growth medium to enhance quicker production. The inoculated medium is maintained under substantial illumination and constant aeration or oxygenation at a temperature of between about 23 and 30°C and preferably at about 24°C. The pH of the medium may be between about 4 and 7 and will typically be about 5. The culture is grown for about 1 to 7 days and usually for about 3 to 5 days depending upon the density required. The medium may optionally include anti-bacterial compounds such as chloramphenicol or rifampicin.

The Trichoderma may be a single strain or a mixture of different strains.

The liquid growth medium will preferably be sterilised before the inoculant is placed in the medium. The volume of the liquid growth medium may be between about 1 litre and 50 litres and is preferably about 8 litres. Naturally, larger or smaller volumes may be used. The process of inoculation will preferably be carried out in a sterile environment.

Naturally, any other method for producing mycelia and conidia containing Trichoderma may be used. The second Trichoderma food source may be selected from molasses, yeast, malt extract and mixtures of any two or more thereof. The diluent may contain between about 1 and 60 gll of molasses. Preferably, it will contain about 20 gll of molasses.

The diluent may contain between about 1 and 8 gll of yeast. Preferably it will contain about 2 gll of yeast. It may further contain between about 0.05 and 2 gll of malt extract and preferably about 1g/-E of malt extract.

The molasses is preferably un-sulphured blackstrap molasses.

Trichoderma growth may be allowed to continue in the aqueous growth medium for a period of between 1 and 7 days or until the density of the Trichoderma is between about 1 ,2x10⁵ and 4x10⁸ spores/mt

The concentration of the malt extract in the liquid growth medium may be between about 10 and 80g/!. The concentration of the peptone may be between about 0.01 and 3g/£. Preferably, the concentration of the malt extract in the liquid growth medium will be about 27 gll and the concentration of the peptone will be about gll.

The ratio between the aqueous growth medium and the diluent may be between about 1 :2 and 1 :4 and is preferably about 1 :3.

The liquid composition of the invention is a stable composition which contains live Trichoderma spores. The composition has a stable shelf-life of up to 12 months if it is stored in a cool area out of contact with direct sunlight or ultraviolet light. Tests carried out 6 months' and 12 months' after preparation of the composition showed that the concentration of spores and colony forming units per ml was retained.

The composition of the invention can be used to protect commercial crops from soil born pathogens and/or to eradicate fungal infestations under varying conditions of soil, temperature and pH. The invention extends to a liquid Trichoderma composition made by a method as hereinbefore described.

The invention extends, further, to a liquid Trichoderma composition which contains live Trichoderma spores and un-sulphured blackstrap molasses.

According to a further aspect of the invention, there is provided a herbicidal liquid Trichoderma composition the composition including live Trichoderma spores and a herbicide.

The herbicidal liquid Trichoderma composition may be prepared by combining a herbicide with a liquid Trichoderma composition as hereinbefore described.

The herbicide may be Polytrin (active ingredient cypermethrine), Avi-Paration (active ingredient parathion) or Avi-Monocrotophos (active ingredient monocrotophos).

Typically, the composition of the invention will be admixed with an aqueous solution of the herbicide.

The resulting herbicidal-containing composition has the combined effects of the Trichoderma composition and a herbicide.

The invention extends still further to a method of treating a plant infected with a plant disease, the method including the step of treating the plant with a liquid Trichoderma composition as hereinbefore described. The method may include treating the soil around the plant with the composition.

The plant disease may be selected from Fusarium, Powdery mildew, Sclerotinia, Altenaria, Downy mildew, Botrytis, Pythium, Phytophthora, Dollar spot, Rhizoctoria solani and similar infections.

Tests were conducted with strain T 315 (ATCC 20671). The composition was found to control Fusarium wilt in tomato plants infected with Fusarium wilt and the plants produced healthy harvests until the end of their normal life cycle. The composition, in combination with a surfactant, was used as a contact spray to stop powdery mildew infections of tomatoes, apples and grapes. The composition also stopped Sclerotinia infections in dried beans, sunflowers and soya beans and stopped Altenaria infections in potatoes, tomatoes, onions and carrots. The composition, in combination with a surfactant, also controlled Downy mildew on grapes and was used as a contact spray to cure Botrytis infections in grapes. The composition also eradicated Pythium infections in water and soil and stopped and cured Dollar spot infections in golf course greens.

The composition was found to be stable for a longer than five minutes at temperatures as high as 60°C. The composition was also evaluated as a growth stimulant and it was found to increase growth in new stems and leaves in grapes and apples by as much as 40%. At the same time the composition increased the sugar levels^" of the grapes by 3%. During a full growing season, in trial runs, 250 hectares of potatoes, 150 hectares of tomatoes, 8 hectares of apples, 4 hectares of grapes, 10 hectares of carrots, 2 hectares of roses and 8 hectares of strawberries were successfully cultivated using only the Trichoderma composition of the invention as a natural fungicide.

A typical example of a liquid growth medium contains 1 I of deionised water, 27 g of malt extract, 1 g of peptone and has a pH of 5. A diluent may contain 1 I of deionised water/ 20 g of malt extract, 3 g of yeast extract and have a pH of 4.8. A preferred diluent contains 1 { of deionised water 20 g of un-sulfured blackstrap molasses, 2 g of yeast extract and have a pH of 4.8

The invention is now described, by way of example, with reference to the following Examples.

EXAMPLE 1

Preparation of the Liquid Trichoderma Composition

Growth Medium Bacteriological malt extract (216 g) and peptone bacteriological powder (8 g) were dissolved in deionised water (8 I) and the pH was adjusted to 5 with HCI or NaOH. The resulting solution was sterilised by autoclaving at 121°C for 40 minutes.

Starter Solution

Bacteriological malt extract (5,4 g) and peptone bacteriological powder (0,2 g) were dissolved in deionised water (200 ml) and the pH was adjusted to 5 with HCI or NaOH. The resulting solution was sterilised at 121 °C for 15 - 20 minutes and inoculated with Trichoderma from a growth plate, Chloramphenicol was added to deactivate any bacterial infection in the solution.

In another embodiment of the invention the starter solution was directly inoculated with Trichoderma grown in a test tube.

Carrier Medium

Un-sulphured blackstrap molasses (60 g), yeast extract powder (6 g) and bacteriological malt extract (3 g) were dissolved in deionised water (3 I) and the pH was adjusted to 4.8 using HCI or NaOH. The resulting solution was sterilised at 121°C for 30 minutes. This procedure was repeated eight times.

Liquid Trichoderma Composition

The starter solution (200 ml) was added to the growth medium (8 I) to produce an inoculated growth medium and the Trichoderma was allowed to grow for a period of 4 days until a spore density of 3,3 x 10⁷ spores/rr was obtained at a constant temperature of 24°C under constant aeration. A 1 I volume of the resulting spore- containing liquid was added to each of the eight carrier medium solutions. The resulting _. live spore-containing liquid was poured into sterile 1 I storage bottles to produce the liquid Trichoderma composition of the invention.

Standard sterile techniques were used during the preparation of liquid Trichoderma composition. EXAMPLE 2

Application of Liquid Trichoderma Composition to Crops

The liquid Trichoderma composition of the invention, as prepared in Example 1 , was used to treat potatoes, tomatoes, apples, grapes, carrots, strawberries and roses. In each case the composition was diluted with sufficient water preferably with a pH of about 5,0-5,5 for the particular application. For example, a volume of 2£/hectare of the composition, as set out in the examples below indicates that 2 of the liquid Trichoderma composition was diluted with sufficient water for application to an area of 1 hectare.

1. Potatoes

The potato crop was drenched using 2^/hectare two weeks after emergence. Thereafter the plants were sprayed bi-weekly using 800 mf/hectare with a surfactant.

2. Tomatoes

At planting the crop was drenched using 2 /hectare. Thereafter the plants were sprayed bi-weekly using 500m£/hectare with a surfactant.

3. Apples

The apple trees were sprayed using 6ϋ/hectare per month with a surfactant.

4. Grapes

The vines were drenched using 2^/hectare at the beginning of the season. Thereafter the plants were sprayed weekly using 2{_^/hectare with a surfactant.

5. Carrots At planting, the crop was drenched using 2£/hectare. Thereafter the plants were sprayed bi-weekly using δOOmϋ/hectare with a surfactant.

6. Strawberries

At planting the crop was drenched using 2ϋ/hectare. Thereafter the plants were sprayed bi-weekly using 500ml!/hectare with a surfactant.

Roses

The roses were drenched using 2^/hectare at the beginning of the season. Thereafter the plants were sprayed bi-weekly using 1 ,5^/hectare with a surfactant.

It is an advantage of the invention illustrated that the composition can also be applied as a seed coating to protect seeds from soil born diseases. The composition can be sprayed, with or without a surfactant, onto the leaves and fruit of plants to protect the plants from pathogens. It can be drenched into the soil by any suitable irrigation method to protect the roots and stems of plants against fungal attacks or to eradicate fungal infection. It is a further advantage of the invention illustrated that the composition is a "natural" or "organic" composition which contains no harmful chemicals or other substances.

It is a further advantage of the invention illustrated that a herbicide can be added to the composition without killing the spores present in the composition. The resulting composition accordingly has the properties of the original composition and has, in addition, herbicidal properties. The Applicant has also found that, where the composition of the invention is used as a natural fungicide, plants were able to use available soil minerals more efficiently when compared with plants treated with chemical fungicides. A comparison of the percentages of various elements present in tomato and potato plants treated with the composition of the invention (labelled "Trykocide") and the percentages of the same elements in plants, treated with conventional chemical fungicides are set out in Tables 1 and 2. TABLE 1

TABLE 2

Claims

CLAIMS:

1. A method of producing a liquid Trichoderma composition, the method including the steps of growing Trichoderma on a growth medium; transferring the Trichoderma from the growth medium to an aqueous growth medium which contains a first Trichoderma food source and allowing Trichoderma growth to continue; and diluting the aqueous growth medium with a diluent which contains a second Trichoderma food source.

2. A method as claimed in Claim 1 , in which the first Trichoderma food source is selected from malt extract, peptone and mixtures thereof.

3. A method as claimed in Claim 1 or claimed 2, in which the second Trichoderma food source is selected from molasses, yeast, malt extract and mixtures of any two or more thereof.

4. A method as claimed in Claim 3, in which the diluent contains between 1 and 60g/ϋ of molasses.

5. A method as claimed in Claim 4, in which the diluent contains about 20g/l of molasses.

6. A method as claimed in any one of Claims 3 - 5 inclusive, in which the diluent contains between 1 and 8g/l of yeast.

7. A method as claimed in Claim 6, in which the diluent contains about 2gll of yeast.

8. A method as claimed in any one of Claims 3 - 7 inclusive, in which the diluent contains between 0.05 and 2g/l of malt extract.

9. A method as claimed in Claim 8, in which the diluent contains about 1g/ϋ of malt extract.

10. A method as claimed in any one of Claims 3 - 9 inclusive, in which the molasses is un-sulphured blackstrap molasses.

11. A method as claimed in any one of the preceding claims, in which Trichoderma growth is allowed to continue in the aqueous growth medium for a period of between 1 and 7 days.

12. A method as claimed in any one of the preceding claims, in which Trichoderma growth is allowed to continue in the aqueous growth medium until the density of the Trichoderma is between 1 ,2x10⁵ and 4x10⁸ spores/mt

13. A method as claimed in any one of Claims 2 to 12 inclusive, in which the concentration of the malt extract in the liquid growth medium is between 10 and 80g/ϋ.

14. A method as claimed in any one of claims 2 - 13 inclusive, in which the concentration of the peptone is between 0.01 and 3g/

15. A method as claimed in Claim 13 or Claim 14, in which the concentration of the malt extract in the liquid growth medium is about 27g/L

16. A method as claimed in Claim 14 or Claim 15, in which the concentration of the peptone is about 1g/L

17. A method as claimed in any one of the preceding claims, in which the volumetric ratio between the aqueous growth medium and the diluent is 1 :3.

18. A liquid Trichoderma composition made by a method as claimed in any one of Claims 1 - 17 inclusive.

19. A liquid Trichoderma composition which contains live Trichoderma spores and un-sulphured blackstrap molasses.

20. A herbicidal liquid Trichoderma composition, the composition including live Trichoderma spores and a herbicide.

21. A herbicidal liquid Trichoderma composition as claimed in Claim 20, in which the herbicide is selected from cypermethrin, parathion and monocrotophos.

22. A method of treating a plant infected with a plant disease, the method including the step of treating the plant with a liquid Trichoderma composition as claimed in any one of Claims 18 to 21.

23. A method as claimed in Claim 22, in which the plant disease is selected from Fusarium, Powdery mildew, Sclerotinia, Altenaria, Downy mildew, Botrytis, Pythium, Phytophthora, Dollar spot and Rhizoctoria solani.

24. A method as claimed in Claim 1 or Claim 22, substantially as herein described and illustrated.

25. A liquid Trichoderma composition as claimed in Claim 18 or Claim 19 or Claim 20, substantially as herein described and illustrated.

26. A new method of producing a composition, a new method of treating a plant or a new composition, substantially as herein described.