WO2010064889A1 - Composition for biological control of plant diseases - Google Patents
Composition for biological control of plant diseases Download PDFInfo
- Publication number
- WO2010064889A1 WO2010064889A1 PCT/MY2009/000086 MY2009000086W WO2010064889A1 WO 2010064889 A1 WO2010064889 A1 WO 2010064889A1 MY 2009000086 W MY2009000086 W MY 2009000086W WO 2010064889 A1 WO2010064889 A1 WO 2010064889A1
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- WIPO (PCT)
- Prior art keywords
- composition
- vermiculite
- trichoderma
- methyl cellulose
- kaolin
- Prior art date
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
- A01N63/38—Trichoderma
Definitions
- the present invention relates to a composition for biological control and prevention of plant fungal diseases, particularly a composition that incorporates Trichoderma viride and Trichoderma harzianum as biocontrol agents.
- Rhizoctonia solani, Phytopthora spp., Pythium spp. and a host of other soil-borne pathogens collectively contribute to commercial crop losses amounting to millions every year. Diseases such as Fusarium. wilt, Pythium blight and damping-off can wipe out an entire planting before it even comes to maturity.
- Intervention practices usually focus on taking out the pathogen after its effects become apparent. Growers often respond to the threat of fungal diseases in crop production with powerful chemicals in an effort to kill off the problem pathogens. ⁇ While it may seem the logical course of action, chemical intervention only serves to make things worse over time. Many chemicals reduce the diversity of soil life even further, risking killing beneficial microbes and increasing resistant pathogens.
- Trichodenna spp. is a naturally occurring soil-borne fungus found in nearly all soils worldwide. Upon contact with roots, Trichoderma will colonize and grow on the root surface. It has been known that these fungi are able to increase the rate of plant growth and development through the production of more robust roots. Moreover, Trichoderma can effectively attack disease-causing pathogens by actively acting as fungal antagonist, attacking (mycoparasitism) other pathogenic fungi through the production of cell wall degrading (hydrolytic) enzymes that destroys the cell walls of the pathogens.
- Trichoderma Although it is known that many species of Trichoderma can be used as potent biocontrol agents for a variety of soil-borne phytopathogenic fungi, the major obstacle to the implementation of these fungi as biological control is the lack in developing and formulating effective and stable preparations, which can be produced commercially and which will be accepted in agricultural systems.
- Another important object of the present invention is to provide an effective and stable formulation for the biological control and prevention of plant fungal diseases; which is suitable for commercial production.
- a composition for biological control and prevention of plant fungal diseases comprising a biocontrol agent, a carrier and an adhesive binder, characterized in that the biocontrol agents are Trichoderma viride and T ⁇ choderma harzianum, the carriers are vermiculite and kaolin and the adhesive binder is carboxyl methyl cellulose.
- the invention disclosed herein is a composition for the biological control and prevention of plant fungal diseases, which basically comprises a biocontrol agent, a carrier and an adhesive binder.
- the composition is specially formulated so that it is effective, stable and suitable for commercial production.
- the composition comprises T ⁇ choderma viride and Trichoderma harzianum as the biocontrol agents, vermiculite and kaolin as the carriers and carboxyl methyl cellulose (CMC) as the adhesive binder.
- the composition comprises 15.39% - 19.23% mixture of liquid media containing Trichoderma viride and Trichoderma harzianum spores, 66.15% - 69.23% vermiculite, 6.92% - 10% kaolin and 0.38% - 0.77% carboxyl methyl cellulose.
- the most preferred formulation of the invention comprises 15.39% mixture of liquid media containing Trichoderma viride and Trichoderma harzianum spores, 68.85% vermiculite, 7.69% kaolin and 0.38% carboxyl methyl cellulose.
- the remaining of the composition is sterilized distilled water coming from the liquid media and CMC solution, which ranges from 3.85% - 7.69%.
- the ratio of liquid medium containing Trichoderma viride to liquid medium containing Trichoderma harzianum is equal (1:1). While the Colony Forming Unit (CFU) of Trichoderma viride and Trichoderma harzianum in the composition is at least 1,0 x 10 7 spores per gram dry weight.
- CFU Colony Forming Unit
- Trichoderma viride and Trichoderma harzianum were chosen as fungal biocontrol agents for many reasons. Primarily, they are known for their numerous mechanisms for attacking other fungi and enhancing plant and root growth such as mycoparasitism, antibiosis, competition for nutrients or space, tolerance to stress through enhanced root and plant development, solubilization and sequestration of inorganic nutrients, induced resistance and inactivation of pathogen's enzymes.
- diseases which they are effective in controlling and treating are diseases that are caused by Fusarium spp., Phytopihora spp., Phytium spp., VerticulHum spp., Rhizoctonia solani, Sclerotia rolfsii, Botrytis spp. and Chondroster ⁇ eun purpureum,
- This invention emphasizes particularly on the formulation of Trich ⁇ derma viride and Trichoderma harzianum with vermiculite, kaolin and carboxyl methyl cellulose.
- Vermiculite and kaolin are porous carriers, wMcfi are capable of supporting fungal growth and are compatible with the plant environment.
- the superfine grade vermiculite is preferred because of its liquid retention ability. 1 L of superfine grade vermiculite can retain up to 457 ml of water before drainage occurs. Vermiculite and kaolin are excellent for the biocontrol composition because they aid aeration, improve moisture retention and contribute a number of minor elements such as potassium and magnesium.
- This biocontrol composition may be applied to various types of plants such as various vegetables, ornamental crops, trees, shrubs, turf grasses, fruit crops, tobacco and plantation crops.
- the composition is particularly excellent for crops such as oil palm, rubber, sugarcane, cocoa, durian, ginger, tobacco, jatropha and kenaf.
- Application is done, preferably at an early stage i.e. during seed stage. It is during this early part of the plant life cycle, which is when the root system is being established and when the plant is most vulnerable to soilborne fungal diseases.
- the biocontrol material may be applied to and/or mixed with the soil or soil-less mix in which plants to be protected by the biocontrol agent will be grown. Alternatively, the biocontrol material may be suspended in water or other liquid and applied to the soil as a drench. In another alternative embodiment, plant seeds may be coated with the biocontrol material.
- Trichoderma viride and Trichoderma harzianum are first grown in culture and caused to sporulate. Cultures are ' maintained on media suitable for sustaining growth under appropriate culture conditions. The ability to grow Trichoderma viride and Trichoderma harzianum is within the knowledge and ability of a person having ordinary skill in the art. Incubation of culture is continued until large quantities of spores are produced. The wet biomass is then mixed with the carriers i.e. vermicu ⁇ ite and kaolin and the adhesive binder i.e. carboxyl methyl cellulose. The carriers and adhesive binder are preferably pretreated to reduce the l&v& ⁇ of contaminating microorganisms. Pretreating may be simply done by autoclaving or for bulk quantity, sterilizing using gamma radiation of 25 kGy.
- Potato Dextrose Agar was used as the nutrient medium for growing Trichoderma viride and Trichoderma harzianum. Inoculum from stock culture was placed at the center of PDA plate. Each strain were duplicated and left for 5 to 7 days.
- Liquid medium molasses media
- Liquid medium was prepared by mixing 5 g of yeast extract and 30 ml of molasses in 1000 ml of distilled water. The liquid mixture was heated for 10 - 15 minutes and autoclaved at 121 0 C for 15 minutes. 1 ml of culture media was then inoculated into 1 L flasks containing 200 ml of liquid media.
- the inoculated flasks were then placed on a shaker and the rotation period was set at 150 rpm for 5 days in which mycelium appears by this time.
- the carriers, vermiculite and kaolin were autoclaved at 121 0 C for 20 minutes and allowed to cool at room temperature before being mixed with the sporulated liquid media.
- Packaging materials were also autoclaved.
- Surface area of a rotary machine was sterilized with 90% ethanol.
- the sterilized solid substrate, vermiculite and kaolin were poured into the rotary machine and mixed thoroughly. Equal ratio of liquid media containing Trichoderma viride and Trichoderma harzianum spores were then poured into the mixture, followed by sterilized carb ⁇ xyl methyl cellulose (CMC) solution.
- CMC carb ⁇ xyl methyl cellulose
- CMC solution was prepared by dissolving 5 g of CMC in 100 ml distilled water; heating the solution on a hot plate for 30 minutes; autoclaving the solution at 121 0 C for 20 minutes; and cooling the solution at room temperature. The whole mixture was mixed well for a couple of minutes. The moisture content of the final product is in the range of 10-30%.
- the preferred formulation for the biological control composition is as shown in Table 1 below.
- the formulation is ideal for maintaining an optimum moisture level of less than 30% and ensuring even distribution of the fungal spores in the final product.
- Table 1 Formulation of Biological Control Composition Viability Test - 100 ml of Tween 80 was added to 10 g of product sample and stirred. The mixture was sonicated for 4 minutes. 1 ml was taken into a sterilized tube. 9 ml of Tween 80 was added and the mixture was stirred. 1 ml of the mixture was then put onto a petri dish (PDA) and results were observed. The result of the viability test is as shown in Table 2.
- Viability test was also done for products that were kept for at least a year.
- the results as in Table 3 show that although some batches had contamination, most batches were still viable after storage of at least one year.
Abstract
A composition for controlling and preventing plant fungal diseases, which comprises a biocontrol agent, a carrier and an adhesive binder. The specially formulated composition incorporates Trichoderma viride and Trichoderma harzianum as the biocontrol agents, vermiculite and kaolin as the carriers and carboxyl methyl cellulose as the adhesive binder.
Description
Composition for Biological Control of Plant Diseases
1. Technical Field of the Invention
The present invention relates to a composition for biological control and prevention of plant fungal diseases, particularly a composition that incorporates Trichoderma viride and Trichoderma harzianum as biocontrol agents.
2. Background of the Invention
Fungal diseases are a constant threat to crop production. Fusarium spp.,
Rhizoctonia solani, Phytopthora spp., Pythium spp. and a host of other soil-borne pathogens collectively contribute to commercial crop losses amounting to millions every year. Diseases such as Fusarium. wilt, Pythium blight and damping-off can wipe out an entire planting before it even comes to maturity.
Intervention practices usually focus on taking out the pathogen after its effects become apparent. Growers often respond to the threat of fungal diseases in crop production with powerful chemicals in an effort to kill off the problem pathogens. ■ While it may seem the logical course of action, chemical intervention only serves to make things worse over time. Many chemicals reduce the diversity of soil life even further, risking killing beneficial microbes and increasing resistant pathogens.
The general principle toward plant disease prevention is to add beneficial soil organisms to improve the soil life. These beneficial organisms will naturally suppress disease through competition, antagonism and direct feeding on pathogenic fungi and bacteria.
Trichodenna spp. is a naturally occurring soil-borne fungus found in nearly all soils worldwide. Upon contact with roots, Trichoderma will colonize and grow on the root surface. It has been known that these fungi are able to increase the rate of plant growth and development through the production of more robust roots. Moreover, Trichoderma can effectively attack disease-causing pathogens by actively acting as fungal antagonist, attacking (mycoparasitism) other pathogenic fungi through the production of cell wall degrading (hydrolytic) enzymes that destroys the cell walls of the pathogens.
Although it is known that many species of Trichoderma can be used as potent biocontrol agents for a variety of soil-borne phytopathogenic fungi, the major obstacle to the implementation of these fungi as biological control is the lack in developing and formulating effective and stable preparations, which can be produced commercially and which will be accepted in agricultural systems.
3. Summary of the Invention
It is a primary object of the present invention to enhance plant health through soilborne biological activity.
It is also an object of the present invention to provide a composition for the biological control and prevention of plant fungal diseases.
Another important object of the present invention is to provide an effective and stable formulation for the biological control and prevention of plant fungal diseases; which is suitable for commercial production.
These and other objects of the present invention are achieved by,
A composition for biological control and prevention of plant fungal diseases comprising a biocontrol agent, a carrier and an adhesive binder, characterized in that the biocontrol agents are Trichoderma viride and Tήchoderma harzianum, the carriers are vermiculite and kaolin and the adhesive binder is carboxyl methyl cellulose.
4. Detailed Description of the Invention
The invention disclosed herein is a composition for the biological control and prevention of plant fungal diseases, which basically comprises a biocontrol agent, a carrier and an adhesive binder. The composition is specially formulated so that it is effective, stable and suitable for commercial production. Specifically, the composition comprises Tήchoderma viride and Trichoderma harzianum as the biocontrol agents, vermiculite and kaolin as the carriers and carboxyl methyl cellulose (CMC) as the adhesive binder. More specifically, the composition comprises 15.39% - 19.23% mixture of liquid media containing Trichoderma viride and Trichoderma harzianum spores, 66.15% - 69.23% vermiculite, 6.92% - 10% kaolin and 0.38% - 0.77% carboxyl methyl cellulose. The most preferred formulation of the invention comprises 15.39% mixture of liquid media containing Trichoderma viride and Trichoderma harzianum spores, 68.85% vermiculite, 7.69% kaolin and 0.38% carboxyl methyl cellulose. The remaining of the composition is sterilized distilled water coming from the liquid media and CMC solution, which ranges from 3.85% - 7.69%. The ratio of liquid medium containing Trichoderma viride to liquid medium containing Trichoderma harzianum is equal (1:1). While the Colony Forming Unit (CFU) of Trichoderma viride and Trichoderma harzianum in the composition is at least 1,0 x 107 spores per gram dry weight.
Trichoderma viride and Trichoderma harzianum were chosen as fungal biocontrol agents for many reasons. Primarily, they are known for their numerous mechanisms for attacking other fungi and enhancing plant and root growth such
as mycoparasitism, antibiosis, competition for nutrients or space, tolerance to stress through enhanced root and plant development, solubilization and sequestration of inorganic nutrients, induced resistance and inactivation of pathogen's enzymes. Among the diseases which they are effective in controlling and treating are diseases that are caused by Fusarium spp., Phytopihora spp., Phytium spp., VerticulHum spp., Rhizoctonia solani, Sclerotia rolfsii, Botrytis spp. and Chondrosterβeun purpureum,
This invention emphasizes particularly on the formulation of Trichσderma viride and Trichoderma harzianum with vermiculite, kaolin and carboxyl methyl cellulose. Vermiculite and kaolin are porous carriers, wMcfi are capable of supporting fungal growth and are compatible with the plant environment. In the present invention, the superfine grade vermiculite is preferred because of its liquid retention ability. 1 L of superfine grade vermiculite can retain up to 457 ml of water before drainage occurs. Vermiculite and kaolin are excellent for the biocontrol composition because they aid aeration, improve moisture retention and contribute a number of minor elements such as potassium and magnesium. We have found that correct mixing of vermiculite, kaolin, carboxyl methyl cellulose and Trichoderma spores is important in order to maintain less than 30% moisture level and ensure even distribution of fungal spores in the final product. Moreover, since the components of the composition are mainly of natural ingredients, hence the biocontrol composition is compatible with and does not affect common pesticides and fertilizers used on crops.
This biocontrol composition, may be applied to various types of plants such as various vegetables, ornamental crops, trees, shrubs, turf grasses, fruit crops, tobacco and plantation crops. The composition is particularly excellent for crops such as oil palm, rubber, sugarcane, cocoa, durian, ginger, tobacco, jatropha and kenaf. Application is done, preferably at an early stage i.e. during seed stage. It is during this early part of the plant life cycle, which is when the root system is being established and when the plant is most vulnerable to soilborne fungal
diseases. The biocontrol material may be applied to and/or mixed with the soil or soil-less mix in which plants to be protected by the biocontrol agent will be grown. Alternatively, the biocontrol material may be suspended in water or other liquid and applied to the soil as a drench. In another alternative embodiment, plant seeds may be coated with the biocontrol material.
For preparation of the product of this invention, Trichoderma viride and Trichoderma harzianum are first grown in culture and caused to sporulate. Cultures are ' maintained on media suitable for sustaining growth under appropriate culture conditions. The ability to grow Trichoderma viride and Trichoderma harzianum is within the knowledge and ability of a person having ordinary skill in the art. Incubation of culture is continued until large quantities of spores are produced. The wet biomass is then mixed with the carriers i.e. vermicuϊite and kaolin and the adhesive binder i.e. carboxyl methyl cellulose. The carriers and adhesive binder are preferably pretreated to reduce the l&v&ϊ of contaminating microorganisms. Pretreating may be simply done by autoclaving or for bulk quantity, sterilizing using gamma radiation of 25 kGy.
The following examples are intended only to further illustrate the invention and are not intended to limit the scope of the invention, which is defined by the claims.
Preparation of Biological Control Composition
Potato Dextrose Agar (PDA) was used as the nutrient medium for growing Trichoderma viride and Trichoderma harzianum. Inoculum from stock culture was placed at the center of PDA plate. Each strain were duplicated and left for 5 to 7 days. Liquid medium (molasses media) was prepared by mixing 5 g of yeast extract and 30 ml of molasses in 1000 ml of distilled water. The liquid mixture was heated for 10 - 15 minutes and autoclaved at 1210C for 15 minutes. 1 ml of culture media was then inoculated into 1 L flasks containing 200 ml of liquid
media. The inoculated flasks were then placed on a shaker and the rotation period was set at 150 rpm for 5 days in which mycelium appears by this time. The carriers, vermiculite and kaolin were autoclaved at 1210C for 20 minutes and allowed to cool at room temperature before being mixed with the sporulated liquid media. Packaging materials were also autoclaved. Surface area of a rotary machine was sterilized with 90% ethanol. The sterilized solid substrate, vermiculite and kaolin were poured into the rotary machine and mixed thoroughly. Equal ratio of liquid media containing Trichoderma viride and Trichoderma harzianum spores were then poured into the mixture, followed by sterilized carbαxyl methyl cellulose (CMC) solution. CMC solution was prepared by dissolving 5 g of CMC in 100 ml distilled water; heating the solution on a hot plate for 30 minutes; autoclaving the solution at 1210C for 20 minutes; and cooling the solution at room temperature. The whole mixture was mixed well for a couple of minutes. The moisture content of the final product is in the range of 10-30%. Although the example given is based on a smaller scale by using a shaker, large-scale production can be carried out by using a bioreactor in which a person having ordinary skill in the art would optimize the parameters of the reactor in terms of temperature, rotation, pH and dissolved oxygen.
Formulation of Biological Control Composition
The preferred formulation for the biological control composition is as shown in Table 1 below. The formulation is ideal for maintaining an optimum moisture level of less than 30% and ensuring even distribution of the fungal spores in the final product.
Table 1: Formulation of Biological Control Composition Viability Test - 100 ml of Tween 80 was added to 10 g of product sample and stirred. The mixture was sonicated for 4 minutes. 1 ml was taken into a sterilized tube. 9 ml of Tween 80 was added and the mixture was stirred. 1 ml of the mixture was then put onto a petri dish (PDA) and results were observed. The result of the viability test is as shown in Table 2.
Table 2: Viability Test on New Batches
Viability test was also done for products that were kept for at least a year. The results as in Table 3 show that although some batches had contamination, most batches were still viable after storage of at least one year.
Table 3: Viability Test on Stored Batches
While particular embodiments of the subject invention have been described, it will be obvious to those skilled in the art that various changes and modifications to the subject invention can be made without departing from the scope of the invention. It is intended to cover, in the appended claims, all such modifications that are within the scope of this invention.
Claims
1. A composition for biological control and prevention of plant fungal diseases comprising a biocontrol agent, a carrier and an adhesive binder, characterized in that the biocontrol agents are Tήchoderma viride and Trichoderma harzianum, the carriers are vermiculite and kaolin and the adhesive binder is carboxyl methyl cellulose.
2. A composition as claimed in claim 1, wherein the composition comprises 15.39% - 19.23% mixture of liquid media containing Trichoderma viride and Trichoderma harzianum spores, 66.15% - 69.23% vermiculite, 6.92% - 10% kaolin and 0.38% - 0.77% carboxyl methyl cellulose.
3. A composition as claimed in claim 2, wherein the composition preferably comprises 15.39% mixture of liquid media containing Trichoderma viride and Trichoderma harzianum spores, 68.85% vermiculite, 7.69% kaolin and 0.38% carboxyl methyl cellulose.
4. A composition as claimed in any one of claims 1 to 3, wherein the remaining of the composition is sterilized distilled water coining from the liquid media and carboxyl methyl cellulose solution, ranging from 3.85% - 7.69%.
5. A composition as claimed in any one of claims 1 to 4, wherein the ratio of liquid medium containing Trichoderma viride to liquid medium containing
Trichoderma harzianum is equal (1:1).
6. A composition as claimed iα any one of claims 1 to 5, wherein the Colony Forming Unit ( CFU ) of Trichoderma viride and Trichoderma harzianum is at least 1.0 x 107 spores per gram (dry weight).
7. A composition as claimed in any one of claims 1 to 6, wherein superfine grade vermiculite is used in the composition.
8. A composition as claimed in any one of claims 1 to 7, wherein the moisture content of the composition is less than 30%.
9. A composition as claimed iα claim 8, wherein the moisture content of the composition is in the range of 10-30%.
10. Use of a composition as claimed in any one of the preceding claims for biological control and prevention of plant fungal diseases.
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MYPI20084926A MY146819A (en) | 2008-12-04 | 2008-12-04 | Composition for biological control of plant diseases |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103409327A (en) * | 2013-08-23 | 2013-11-27 | 中国热带农业科学院环境与植物保护研究所 | Method for producing green trichoderma viride through liquid/solid double-phase fermentation |
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JP2004123606A (en) * | 2002-10-02 | 2004-04-22 | Kumiai Chem Ind Co Ltd | Wettable composition for agriculture, method for producing the same, and preservation method for the same |
US20040151699A1 (en) * | 2002-07-26 | 2004-08-05 | Salinas Mario Reyes | Bactericidal, bacteriostatic and fungicidal composition, comprising two or more live species of trichoderma and its preparation procedure, and a composition based on latex for a pruning cicatrizing that includes a latex base and a composition of two or more live species of trichoderma |
WO2005095579A1 (en) * | 2004-03-31 | 2005-10-13 | Danisco A/S | Process |
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US5849320A (en) * | 1996-06-13 | 1998-12-15 | Novartis Corporation | Insecticidal seed coating |
US20040151699A1 (en) * | 2002-07-26 | 2004-08-05 | Salinas Mario Reyes | Bactericidal, bacteriostatic and fungicidal composition, comprising two or more live species of trichoderma and its preparation procedure, and a composition based on latex for a pruning cicatrizing that includes a latex base and a composition of two or more live species of trichoderma |
US7422737B1 (en) * | 2002-09-05 | 2008-09-09 | Yissam Research Development Company of the Hebrew University of Jerusalem | Porous freeze-dried hydrocolloid beads containing viable microorganisms for biological control |
JP2004123606A (en) * | 2002-10-02 | 2004-04-22 | Kumiai Chem Ind Co Ltd | Wettable composition for agriculture, method for producing the same, and preservation method for the same |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103409327A (en) * | 2013-08-23 | 2013-11-27 | 中国热带农业科学院环境与植物保护研究所 | Method for producing green trichoderma viride through liquid/solid double-phase fermentation |
CN103409327B (en) * | 2013-08-23 | 2015-03-25 | 中国热带农业科学院环境与植物保护研究所 | Method for producing green trichoderma viride through liquid/solid double-phase fermentation |
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