WO1998031220A1

WO1998031220A1 - The formulation and utilization of a fungicidal product

Info

Publication number: WO1998031220A1
Application number: PCT/SE1998/000094
Authority: WO
Inventors: Jacques Ricard; Thomas Ricard
Original assignee: Jacques Ricard; Thomas Ricard
Priority date: 1997-01-21
Filing date: 1998-01-21
Publication date: 1998-07-23
Also published as: SE508838C2; SE9700189L; SE9700189D0; AU5787898A

Abstract

The present invention relates to a formulation with fungicidal activity, a method of manufacturing the fungicidal formulation as well as its use. The formulation contains finely ground dry powdered propagule containing material produced from sterilized cereal materials inoculated with mycoparasites and also finely ground sifted pulverized sterilized clay material and optionally a material improving consistency.

Description

The Formulation and Utilization of a Fungicidal Product

The present invention relates to a formulation with fungicidal activity, a method of manufacturing the fungicidal formulation and use of the formulation.

During the cultivation of plants and fruit, such as strawberries, tomatoes, cucumber, etc. attacks of gray mold (Botrytis cinerea) are a large and costly problem. To prevent and control these attacks of fungi, the plants respectively the fruits are sprayed with different fungicidal compounds, which in most of the cases consist of chemical substances. Synthetic chemicals for this purpose are an environmental drawback for the ecological system closest to the treated area, but in the long run also the global system is loaded by the unaltered products as well as residual products of such chemical compounds that have been broken down or been converted in some other way.

Especially strawberries have a short shelf life and therefore have to be treated soon before the harvest. This involves a special problem, because addition of chemical compounds may be directly harmful for the consumer of the fruit .

The present invention solves the above mentioned problems by the new effective formulation, which is an effective controlling agent of these damaging fungi, at the same time as the environmental impact is minimized, since the present formulation does not contain the synthetic substances that cause the mentioned side effects. Accordingly, it is an object of the present invention to provide a formulation with fungicidal activity, which includes finely ground dried pulverized propagule material manufactured from sterilized grain material, which has been inoculated with mycoparasites, mixed with finely ground, sifted pulverized sterilized clay material and possibly consistency improving agent. Another object of the present invention is to provide a method of manufacturing a formulation with fungicidal activity. The method provided by the invention comprises the following steps: a) inoculation of mycoparasites on sterilized grain material in the presence of water, b) incubation at temperature suitable for mycoparasites to form propagules, c) drying of the material when it is overgrown by conidia, d) grinding of the mentioned material, and e) mixing of the material with finely ground sterilized and sifted clay material to a powdery mixture, which is suitable for blending with water. Consistency improving agent may be added to the formulation.

A further object of the present invention is the use of the fungicidal formulation for the manufacture of aqueous suspensions, which are sprayed, sprinkled or atomized on plants, fruits or pasteurized plant substrate. A preferred object is the use of the formulation according to the invention on strawberry plants,

A preferred embodiment of the present formulation comprises finely ground grain, which has been inoculated with Trichoderma polysporum ATCC 20475, Trichoderma harzianum ATCC 20476 and/or Scytalidium spp. ATCC 16675, sifted and dried sterilized silicon containing clay materials, preferably Arctic clay. The clay materials have a preferred particle size of less or equal to 500 μm. The preferred clay materials have a pH within the range of 3 to 6,5, preferably within the range of 4 to 6, most preferably within the range of 4,5 to 5.0.

In an another preferred formulation, the contents of Trichoderma flour and/or Scytalidium flour in proportion to the clay materials are in the range of 10-30 % to 70-90 %. A preferred method of manufacturing the fungicidal formulation comprises the following steps: a) inoculation of mycoparasites, preferably Trichoderma polysporum ATCC 20475, Trichoderma harzianum ATCC 20476 and/or Scytalidium spp. ATCC 16675, on sterilized grain material in the presence of water; b) incubation at temperature suitable for the formation of propagules by the mycoparasites; c) drying of the material when it is overgrown by conidia and/or arthrospores ; d) grinding of the mentioned material and sifting to fractions larger or smaller than 350 μm; e) mixing of the mentioned material with finely ground sterilized and sifted clay materials, which have been sifted to a particle size less than or equal to 500 μm; f) mixing of the components to a powdery mix, which is suitable for mixing in water; g) possible addition of consistency improving agent.

Preferred uses of the formulation according to the invention are spraying, sprinkling or atomizing of an aqueous suspension of the formulation on plants, such as salads, herbal plants, potted plants etc., on fruits such as strawberries, tomatoes, cucumber etc., or on plant substrates, such as pasteurized peat or mineral wool. The present invention results, as mentioned above, in that the stress on the environment may be decreased, but also results in that the formulations of Trichoderma and/or Scytalidium have obtained a considerably improved storage stability with almost undiminished activity during a long storage time.

Earlier formulations of Trichoderma and/or Scytalidium have lost up to 90 % per year of their original activity at storage in room temperature.

Modern storage methods of microorganisms comprise freeze drying or treatment with liquid nitrogen. These methods are both costly and not suitable to use agricul- tural ly .

By mixing flour of Trichoderma and/or Scytalidium with silicon containing clay materials, the mentioned storage stability is achieved, but the clay materials also function as a binding agent, for example for the production of pellets .- Earlier formulations contain chemicals which require o a higher temperature for the production of pellets >50 C. o

Fungal spores die at temperatures exceeding 50 C, this is also the case with Trichoderma spp. and Scytalidium spp. respectively.

As mentioned earlier the formulations according to the present invention are especially suitable for fruits and vegetables with a short shelf life, but also for pasteurized plant substrates, mainly for two reasons. Firstly, Trichoderma polysporum ATCC 20475, Trichoderma harzianum ATCC 204476 and Scytalidium spp ATCC 16675 stop growing at o temperatures over 30 C, which means that there is a safety o margin of 5 to 7 C to the human body temperature, and therefore, they do not cause mycoses. Secondly, sterile clays are completely acceptable from all environmental and immune aspects .

Fruits which are suitable to be treated with the formulation according to the invention are for example strawberries, cucumbers, tomatoes etc. The formulation according to the invention is also suitable to be used on different types of plants, such as salads, herbal plants or potted plants. To get a successful cultivation result the formulation is also suitable for pasteurized plant substrates such as pasteurized peat or mineral wool. The pasteurized plant substrate may be in the form of briquettes, pots or in any other form intended for planting of plants.

The formulation according to the invention is especially suitable to be used against fungal attacks, gray mold was mentioned earlier, but other fungal attacks may also be prevented by the formulation. The following fungal attacks are suitable to be treated with the formulation:

1) gray mold on fruit, fruit trees, conifers, grapevines and grapes;

2) si-lver leaf disease (Chondrostereum purpureum) on fruit trees;

3) mildew on strawberry plants in open fields;

4) potato late blight (Phytophthora infestans) on pota- toes;

5) fungal pathogens in plant substrate (e.g. soil and mineral wool), as Pythium ultimum, Rhizoctonia solani, Fusarium spp., Phytophthora spp., or Verticillium spp.

The formulation according to the invention is suitably dosaged in amounts of 1 to 2 g/plant or 50 to 300 g/m3 plant substrate .

The above mentioned and other preferred embodiments of the invention are defined by the dependent claims.

Example 1

A batch of 0.5 kg grain saturated with Trichoderma harzianum ATCC 20476 was dried to a moisture content of 6 to 9 %. The dry and clumpy mass was first ground in a hammer mill to a particle size of 3 mm,, whereafter the grain was ground to flour in a stone mill.

The Trichoderma flour was sifted to a sift size of 350 μm in a continuous sifter. The finer fractions were tested by growing different dilutions on malt agar and incubation at o room temperature and at 37 C. Further tests were made with o nutrient agar incubated at 37 C.

The agar plates were checked after 24 respectively 36 hours, the colonies were counted for suitable dilutions to amount to at least 10 up to 100 colonies per plate. The CFU/gram value (Colony Forming Unit) for the Trichoderma flour was calculated through these values. After counting of the colonies the plates were incubated for another week to 10 days at room temperature under fluorescent light for development of conidia, to be able to identify the presence of fungi on the plates, especially to identify the presence of unwanted fungi, as Penicillium or Aspergillus spp. Normally no such unwanted species are present on the plates.

The incubated nutrient agar plates show within 24 hours or at the most 72 hours if bacteria colonies are present in the Trichoderma flour.

The pure 10 to 30 weight-% Trichoderma flour, depending on the calculated Trichoderma activity in the flour, was thereafter mixed with 90 to 70 weight-% silicon containing clay, which has been autoclaved and ground fine. The finished powder mix can then be used as is or in the form of pellets. The finished product has a durability that does not get lost as pure Trichoderma flour, which loses ca. 10 % of its CFU/gram value per month at storage.

Example 2

A batch of 0.5 kg grain saturated with Trichoderma polysporum ATCC 20475 was dried to a moisture content of 6 to 9 %. The dry and clumpy mass was first ground in a hammer mill to a particle size of 3 mm, thereafter the grain is ground to flour in a stone mill..

The Trichoderma activity was calculated in the manner described above, whereafter 10 to 30 weight-% of the flour was mixed with 90 to 70 weight-% silicon containing clay, which has been autoclaved and ground fine.

Example 3

Field tests were made with strawberry plants, where suspension of the two Trichoderma formulations according to the invention was applied on the plants when they started blooming. The applications were made when 5-10 %, 10-15 %, 40,%, 60 % and 100 % of the flowers were in bloom. Control tests were made without spraying of fungicidal formulation on the plants. The control test resulted in a harvest of 1233 kg/0.1 ha. Trichoderma formulation was dosaged in the different tests in doses of 500 g/ha and 250 g/ha. The yield of strawberries increased with the Trichoderma formulation. The different doses gave the following result: 500 g/ha gave an increased yield of 23 % and 250 g/ha an increased yield of 14 %.

A test was made with iprodion in a dose of 3.0 1/ha (recommended dosage of the pesticide for control of gray mold on strawberries) and the harvest increased only by 5 % . Thus the tests show that the Trichoderma formulation according to the present invention has increased the harvest of strawberries, also in low doses, both compared to the control test and compared to a conventional compound as iprodion.

Example 4

A batch of 0.5 kg grain saturated with Scytalidium spp. ATCC 16675 was dried to a moisture content of 6 to 9 % . The dry and clumpy mass was first ground in a hammer mill to a particle size of 3 mm, thereafter the grain is ground to flour in a stone mill.

The Scytalidium activity was calculated in the manner described above, where after 10 to 30 weight-% of the flour was mixed with 90 to 70 weight-% silicon containing clay, which has been autoclaved and ground fine.

Example 5

Laboratory tests have shown that Scytalidium spp. ATCC 16675 and its metabolites inhibit respectively control fungal pathogens, which are damaging for plants in vitro and in vivo. Damaging fungal pathogens are e.g. Armillaria me1lea, Lenzites trabea, Graphium spp. Polyporus tomenlosus and Poria carbonica .

The laboratory tests, which have been used to study the effectiveness, have been made according to standardized methods.. In petri dishes the agar was inoculated on different sides of the dish with a fungal pathogen, as Armillaria mellea, Lenzites trabea, Graphium spp., Polyporus tomenlosus and Poria carbonica, respectively with the formulation manufactured according to Example 4. The cultivation was left for a week up to one month depending on the qualities of the fungal pathogen. The spreading of the organisms was studied, also the occurrence of parasitizing and production of metabolites. One found that where the formulations according to Example 4 were inoculated, the fungal pathogens had been inhibited and hyperparasitized.

Further tests were made according to the soil-block technique where a sample (a) of wood e.g. spruce was inoculated with fungal pathogens, as Armillaria mellea, Lenzites trabea, Graphium spp., Polyporus tomenlosus and Poria carbonica. The sample (a) was placed on pasteurized soil in a closed container. On top of the sample (a) was placed a sample (b) earlier smeared with the formulation according to Example 4. The closed container with the two samples (a) and (b) was placed in a growth chamber. After some time the test was evaluated -by measuring the weight reduction of treated sample (b) and comparing this with the weight reduction of a zero-test with untreated sample (b) . The weight reduction is a result of the weight loss that occurs when fungal pathogens break down the cellulose in the sample. The test showed that treated sample (b) had not appreciably lost in weight as compared to untreated sample (b) , which had been broken down by fungal pathogens.

The above examples do not limit the invention but merely make it evident.

Claims

1. A fungicidal formulation comprising finely ground dried pulverized propagule material manufactured on sterilized grain materials, which have been inoculated with mycoparasites, such as Trichoderma polysporum ATCC 20475, Trichoderma harzianum ATCC 20476 and/or Scytalidium spp. ATCC 16675, characterized in that the formulation also contains finely ground, sifted pulverized sterilized clay materials and optionally a consistency improving agent, the proportion of propagule material to clay materials is in the range of 10-30 % : 70-90%.

2. A fungicidal formulation according to claim 1, characterized in that the clay materials have been sifted to a particle size less or equal to 500 ╬╝m, and have a pH within the range of 3 to 6.5, preferably within the range of 4 to 6.

3. A fungicidal formulation according to claim 2, characterized in that the pH of the clay material is within the range of 4.5 to 5.0.

4. A fungicidal formulation according to any of claims 1 to 3, characterized in that the clay materials is silicon containing clays, preferably Arctic clay.

5. A fungicidal formulation according to any of the preceding claims, characterized in that the formulation is available in the form of wettable powder, water dispersible granules or pellets.

6. A method of manufacturing a formulation with fungicidal activity, comprising inoculating sterilized grain materials with mycoparasites, such as Trichoderma polysporum ATCC 20475, Trichoderma harzianum ATCC 20476 and/or Scytalidium spp. ATCC 16675, characterized in that inoculation and incubation of the grain materials is performed at a temperature adapted to the mycoparasites for the formation of propagules in the presence of water, drying the propagules, when the grain materials are overgrown by conidia and/or arthrospores, then grinding the propagule material and mixing it with -finely ground sterilized and sifted clay materials to a blend of powder of the formulation, which is appropriate for mixing in water and consists of between 10 and 30 % of the propagule material and between 70 and 90 % of the clay materials.

7. A method process according to claim 6, characterized by sifting the ground propagule material into fractions larger than respectively smaller than 350 ╬╝m, before mixing it with the clay materials.

8. A method process according to claims 6 and 7, characterized by sifting the clay materials to a particle size less than or equal to 500 ╬╝m, the pH of the clay materials being within the range of 3 to 6.5, preferably within the range of 4 to 6.

9. A method according to claim 8, characterized in that the pH of the clay material is within the range of 4.5 to 5.0.

10. A method process according to any of the preceding claims, characterized by selecting the clay materials from silicon containing clays, preferably Arctic clay.

11. A method according to any of the preceding claims, characterized by manufacturing the fungicidal formulation in forms of wettable powder, water dispersible granules or pellets .

12. Use of a fungicidal formulation according to any of claims 1 to 5, for the manufacture of an aqueous suspension for spraying, sprinkling or atomizing on plants, fruits, trees or pasteurized plant substrates for the treatment or the prevention of fungal attacks.

13. Use according to claim 12, characterized in that the plants comprise strawberry plants, cucumber plants, tomato plants, salad plants or potted plants.

14. Use according to claim 12, characterized in that the fruits comprise strawberries, grapes, cucumbers or tomatoes.

15. Use according to claim 12, characterized in that the pasteurized plant substrates comprise pasteurized peat or mineral wool .

16. Use according to claim 15, characterized in that the pasteurized plant substrates are in the form of briquettes, pots or other forms which are intended for planting plants therein, such as useful plants or potted plants.

17. Use according to any of claims 12 to 16, characterized in that the formulation according to any of claims 1 to 7 is

3 dosed in an amount of 1 to 2 g/plant or 50 to 300 g/m plant substrate .

18. Use of a fungicidal formulation according to any of claims 1 to 5, for the manufacture of an aqueous suspension, for spraying, sprinkling or atomizing on plants, fruits, trees or pasteurized plant substrates for the treatment or the prevention of fungal attacks, such as gray mold (Botrytis cinerea) , silver leaf disease (Chondrostereum purpureum) , mildew, potato late blight (Phytophthora infestans), Pythium ultimum, Rhizoctonia solani, Fusarium spp., Phytophthora spp., or Verticillium spp.