Trichoderma harzianum bioformulation for controlling root and crown rot disease and enhancing plant growth in edible-seeded pumpkin

Abstract

The invention relates to a granular bioformulation containing Trichoderma harzianum (NCBI accession number: MH351669) isolate TRIC8, supplemented with wheat straw and sugar beet molasses, developed to prevent root and crown rot disease caused by seed-borne Fusarium proliferatum in edible-seeded pumpkin used in the production of snacks, confectionery, and desserts, and to promote plant growth.

Description

[0001] DESCRIPTION

[0002] TRICHODERMA HARZIANUM BIOFORMULATION FOR CONTROLLING ROOT AND CROWN ROT DISEASE AND ENHANCING PLANT GROWTH IN EDIBLE- SEEDED PUMPKIN

[0003] Technical Field of the Invention

[0004] The invention relates to a granular bioformulation containing Trichoderma harzianum (NCBI accession number: MH351669) isolate TRIC8, supplemented with wheat straw and sugar beet molasses, developed to prevent root and crown rot disease caused by seed-borne Fusarium proliferatum in edible-seeded pumpkin used in the production of snacks, confectionery, and desserts, and to promote plant growth.

[0005] State of the Art

[0006] Soil-borne fungal diseases are a problem in cultivated plant production and cause significant crop losses. Soil-borne fungal pathogens can survive in soil for many years by constantly adapting to changing soil conditions. Crop rotation, use of resistant cultivars, some fungicides, soil sterilants and solarization are recommended for the control of such pathogens which cause significant economic losses in plants, but they are not successful enough. It is known that soil fumigants cause residue problems on crops, pollute soil, water and air, and adversely affect humans and other living organisms.

[0007] Common fungal diseases in pumpkin cultivation include wilt, root and crown rots, downy mildew, gray mold, powdery mildew, anthracnose and some foliage and fruit spot diseases [1]. The severity of these diseases depends on the virulence of the pathogen, the physical and chemical properties of the growth medium, soil moisture, temperature and pH, nutritional factors, and the susceptibility of the host. The occurrence of the disease is probable when the soil environment is conducive to fungal development but is not favourable for the host. The above-ground symptoms of root rot are the result of a weakened root system and usually manifest as nutrient deficiency, poor growth, chlorosis, wilting, or plant death. Crop rotation is of great importance in preventing soil-borne fungal diseases in edible-seeded pumpkin cultivation. Root rot disease is caused by more than one pathogen in cultivation areas. Considering the cultivation conditions in Turkey, Rhizoctonia spp., Sclerotinia spp., Fusarium spp. are among the common soil-borne fungal species that can negatively affect production. However, soil-borne fungi such as Pythium and Phytophthora, whose zoospores are carried in water, become widespread over time with the increase in humidity in the regions where dry agriculture is switched to irrigated agriculture within the scope of irrigation projects. Soil-borne fungal pathogens, defined as damping-off, wilt, and root and crown rots, cause significant yield losses in the crop. These pathogens can survive in the soil for a long time in the form of resistant spores. The most common symptoms of the disease in plants are the rotting of seeds in the soil before germination, rotting of the crown of developing seedlings, and the lodging of seedlings on the soil surface due to thinning of the crown after emergence. Furthermore, seedlings emerging to the soil surface may show foliage deformations. The roots of diseased seedlings show softening and browning, especially in the bark tissue. In infected fields, patchy damping-off, partial wilting, and sparse emergence are observed. In severe infections, replanting is required in the field. This causes loss of labor and production [2], For edible-seeded pumpkin, the seed-borne fungal pathogen F. proliferatum caused root and crown rot in 51.07% of susceptible cultivar when inoculated on seeds and 43.75% when inoculated on soil. Furthermore, when inoculated from both soil and seed, it causes significant reductions in the number of leaves, shoot length, shoot fresh and dry weight of developing plants.

[0008] Fusarium species are fungi that can adapt to different climatic conditions in the world except Antarctica [3]. Pathogenic Fusarium species exhibit symptoms in the form of wilt or root rot in more than 100 host plants and can survive in the soil for a long time with their resistant spore structures. These fungi of soil and seed origin can remain viable for 2 years in the seed and for many years in the soil in the form of chlamydospores. Chemical control of soil-borne Fusarium species is very difficult, however, crop rotation practice prevents the disease to a significant extent. When crop rotation is not applied in a production area, there may be many negativities such as the increase in the population of soil-borne disease agents, the activation of nematodes, the decrease in the amount of organic matter in the soil, the disruption of the balance of nutrients in the soil, and the increase in toxic residue problems of pesiticides. Solarization, which is a physical method included in integrated control programs against soil-borne pathogens, nematodes and weeds due to its wide spectrum of action and environmental protection, is also used in the control of soil-borne Fusarium species. Solarization is the process of disinfecting the soil with the heat energy of the sun. It is an environmentally friendly method that does not contain toxic materials, is economical, and easy to implement [4], The solarization process usually takes several weeks. This affects the agricultural production calendar and leads to delays. In addition, the treatment is usually effective on the surface. Its effect against pathogens deep in the soil is limited. In addition, seed disinfection with sodium hypochlorite (1%) before sowing is important in the control of soil-borne diseases [2], In addition, fungicides with the active ingredients hymexazole, captan, captan+pencycuron, hydroxyquinoline sulphate, azoxystrobin + metalaxyl-m + fludioxonil, fludioxonil + metalaxyl-m and propamocarb + fosetyl, and fumigants with the active ingredients ethandinitrile, metam potassium and metam sodium are used as soil applications in seedbeds. Chemical control causes water and soil pollution, and the misuse of pesticides disrupts ecosystem balances.

[0009] In order to protect the biological balance that has been disrupted by excessive use of chemicals, research on alternative control methods that protect environmental health is gaining momentum day by day. It has been reported by many researchers that root bacteria, particularly Pseudomonas fluorescens, have the ability to inhibit soil-borne fungal pathogens through mechanisms such as competition with the pathogen, induction of systemic resistance (ISR; Induced Systemic Resistance), and the production capability of siderophores, mycolytic enzymes, and antibiotics [5-6]. In many countries around the world, commercial preparations of Bacillus, Pseudomonas and Streptomyces species are practically available for biological control of plant diseases [7], The most commonly used ones are bacteria such as Bacillus spp., Pseudomonas spp. Streptomyces spp. Agrobacterium radiobacter and fungi such as Trichoderma spp., Candida oleophila, Gliocladium spp. and Coniothyrium minitans, Ampelomyces quisqualis [8]. Trichoderma harzianum Rifai KRL-AG2 (T-22 Planter Box), Trichoderma harzianum Rifai KRL-AG1 (Rootshield Granules), Trichoderma harzianum Rifai Strain T22 (Trianum P), Trichoderma aspellerum strain ICC 012+ Trichoderma gamsii strain ICC 080 (Remeider), Trichoderma viride (Bio-Cure F), Bacillus subtilis IAB / BS03 (Fungisei), Gliocladium virens strain GL21 (Soilgard 12 G), Bacillus amyloliquefaciens B. subtilis) strain QST 713 (Serenade) are used as registered biological fungicides for root diseases in Turkey. Of these, only Trianum P. is applied to cotton seeds, the others are applied to the soil. In the state of the art, the company GreenMax Agro Tech produces a preparation named Trichoderma harzianum Bio Fertilizer Liquid with talc additive as a plant growth promoter. This bioformulation consists of Trichoderma harzianum 1% (w / w), adhesive-carboxymethyl cellulose (CMC, 1%) inactive material (98%, w / w), and talc (63%). Bioformulation is used as seed application (mixed at a rate of 40 g / kg seed and dried in the shade for 24 hours), seedling dipping (2 kg / 50 I water, 10 minutes) and soil application (5 kg bioformulation mixed with 100 kg organic matter and applied to the soil after being kept in the shade for 1 week). Said bioformulation is reported to cause the formation of cold-resistant roots, deep roots in maize and ornamentals and to be effective against B. cinerea.

[0010] Due to reasons such as the limitations and shortcomings of the present technical solutions, the fact that the root rot disease caused by seed-borne Fusarium proliferatum in Cucurbitaceae plants, especially in edible-seeded pumpkin, causes significant reductions in leaf number, shoot length, shoot fresh and dry weight, chemical control methods cause water and soil pollution, improper use of pesticides disrupts ecosystem balances, the solarization process takes a long time and causes delays in the production schedule and the lack of a registered fungicide or biopreparation against the root rot disease caused by F. proliferatum, it has become necessary to make an improvement in the prevention of root rot disease caused by seed-borne Fusarium proliferatum in plants of the Cucurbitaceae family, especially in edible-seeded pumpkin.

[0011] Summary and Objects of the Invention

[0012] The invention describes a granular bioformulation containing Trichoderma harzianum (NCBI accession number: MH351669) isolate TRIC8, supplemented with wheat straw and sugar beet molasses, developed to prevent root rot disease caused by seed-borne Fusarium proliferatum in edible-seeded pumpkin used in the production of snacks, confectionery, and desserts, and to promote plant growth.

[0013] The object of the invention is to prevent the root rot disease caused by seed-borne Fusarium proliferatum in plants of the family Cucurbitaceae, especially in the edible-seeded pumpkin, and to promote plant growth. An object of the invention is to provide a bioformulation which has a low production cost and is easy to produce. Wheat straw and sugar beet molasses contained in the bioformulation of the invention are readily available low-cost ingredients. In addition, bioformulation is applied in smaller quantities because it can be targeted directly to seeds rather than over large areas of agricultural land. There is no need for repeated application. This not only reduces the cost of production but also saves farmers' expenses. In addition, because bioformulation can be applied to seed, the production can be realized at low cost.

[0014] Another object of the invention is to provide a bioformulation that can maintain its viability for a long time. The sugar beet molasses additive contained in said bioformulation enables T. harzianum conidia to easily adhere to the seed and wheat straw, thus maintaining the viability of the bioformulation on the seed for 60 days.

[0015] Description of the Drawings

[0016] Fig. 1. Inhibition rates of colony growth of Fusarium proliferatum at 5 different doses of different bioformulations of Trichoderma harzianum (Doses; a: 0.25%, b: 0.50%, c: 0.75%, d: 1%, e: 2%).

[0017] Fig. 2. Colony growth of Fusarium proliferatum on PDA medium when bioformulations prepared with Trichoderma harzianum (TRIC8) were applied at different doses (A-E: Conidia suspension of antagonist+CMC (TF1), F-J: Talc+CMC (TF2), K-O: Talk+CMC+CaCOs (TF3), P-T: Pesta granule (TF4), U-Y :Trichoderma harzianum (TRIC8) bioformulation (Invention), Z-d: Corn straw (TF6), e: Control). (Gray-black areas represent bioformulations and white areas represent Fusarium proliferatum)

[0018] Fig. 3. Disease severity in seedlings after inoculation of seeds with Fusarium proliferatum followed by coating with the inventive Trichoderma harzianum (TRIC8) bioformulation.

[0019] Detailed Description of the Invention

[0020] The invention relates to a granular bioformulation containing Trichoderma harzianum (NCBI accession number: MH351669) isolate TRIC8, supplemented with wheat straw and sugar beet molasses, developed to prevent root and crown rot disease caused by seed-borne Fusarium proliferatum in edible-seeded pumpkin used in the production of snacks, confectionery, and desserts, and to promote plant growth. The inventive bioformulation prevents seedling root and crown rot by 75.6% and increases seedling root length, shoot length, seedling vigor index, seedling fresh and dry weights in seeds of edible-seeded pumpkin contaminated with F. proliferatum.

[0021] The inventive bioformulation comprises 2.0-3.0% Trichoderma harzianum (NCBI accession no: MH351669) TRIC8 isolate, 37.0-41.0% wheat straw and 57.0-60.0% of the sugar beet molasses diluted with 8-12% distilled water. In an embodiment of the invention, the inventive bioformulation comprises 2.3% Trichoderma harzianum (NCBI accession no: MH351669) TRIC8 isolate, 39.1% wheat straw and 58.6% sugar beet molasses diluted with 10% distilled water.

[0022] The preparation method of the inventive bioformulation comprises the processing steps of:

[0023] i. soaking the ground wheat straw with distilled water, then removing the excess water and adding sugar beet molasses diluted with distilled water,

[0024] ii. sterilizing the resulting mixture in autoclave and adding Trichoderma harzianum TRIC8 isolate from a conidial suspension,

[0025] iii. drying in a sterile environment by mixing every day,

[0026] iv. after the drying process, passing it through sterilized sieves in a sterile environment, taking into sterile jars, and closing the lids with vacuum.

[0027] In another embodiment of the invention, the preparation method of the inventive bioformulation comprises the processing steps of:

[0028] i. soaking ground wheat straw in distilled water at 250-350 ml / 100 g of straw for 1.5- 2.5 hours, removing the excess water, adding sugar beet molasses, diluted to 8- 12% with distilled water, at 140-160 ml / 100 g of straw,

[0029] ii. sterilizing the mixture obtained by autoclaving it 3 times at 121 °C for 20 minutes at 1-day intervals and adding 5-7 ml / 100 g of straw from the conidial suspension of Trichoderma harzianum TRIC8 isolate (1 x 107conidia / ml). iii. drying in a sterile environment at 24-26°C for 4-6 days by stirring daily, iv. after the drying process, passing it through sterilized sieves with a mesh size of 0.5-1.4 mm in a sterile environment, transferring it into sterile jars, and closing the lids with vacuum.

[0030] In one embodiment of the invention, the preparation method of the inventive bioformulation comprises the processing steps of:

[0031] i. soaking ground wheat straw in distilled water at 300 ml / 100g of straw for 2 hours, removing the excess water, adding sugar beet molasses, diluted to 10% with distilled water, at 150 ml / 100 g of straw,

[0032] ii. sterilizing the mixture obtained by autoclaving it 3 times at 121 °C for 20 minutes at 1 -day intervals and adding 6 ml / 100 g of straw from the conidial suspension of Trichoderma harzianum TRIC8 isolate (1 x 107conidia / ml).

[0033] iii. drying in a sterile environment at 25°C for 5 days by stirring daily,

[0034] iv. after the drying process, passing it through sterilized sieves with a mesh size of 1 mm in a sterile environment, transferring it into sterile jars, and closing the lids with vacuum.

[0035] While preparing the inventive bioformulation, ground wheat straw was soaked with distilled water (300 ml / 100 g of straw) for 2 hours, excess water was removed and sugar beet molasses (10%) was added (150 ml / 100 g of straw). The mixture obtained was sterilized by autoclaving it 3 times at 121 °C for 20 minutes at 1 -day intervals and 6 ml / 100 g of straw from the conidial suspension of Trichoderma harzianum TRIC8 isolate (1 x 107conidia / ml) was added. Then, it was dried in a sterile environment at 25°C for 5 days by stirring. This process showed that the antagonist remained viable. After the drying process, it is passed through sterilized sieves with a mesh size of 1 mm in a sterile environment, transferred it into sterile jars, and its lids are closed with vacuum. The shelf life of the bioformulation is 150 days at +4 °C.

[0036] In order to control root rot disease and to strengthen plant growth in edible-seeded pumpkin, clean and mechanically undamaged seeds are slightly moistened (1 / 3 surface of the seed is wetted) and placed in a clean area. The bioformulation is sprinkled on the seeds at a dose of 1 g / 100 g of seed, mixed until the surface is completely covered with bioformulation, and dried in a clean environment for 15 minutes. Firstly, the ability of the inventive bioformulation to inhibit the colony growth of pathogenic Fusarium proliferatum, which causes root and crown rot disease in edible-seeded pumpkin (Cucurbita pepo) plants was determined. For this purpose, 6 different bioformulations were made with T. harzianum TRIC8 isolate and these were mixed with Potato Dextrose medium at 5 different doses. The trials were conducted with 5 replications. Among the 6 bioformulations, the bioformulation prepared with wheat (Figures 1 and 2) was found to inhibit the colony growth of the pathogen at the highest rate. Among the doses, 1% and 2% doses of the inventive bioformulation were found to have the highest ability to inhibit the colony growth of the pathogen with 94.13% and 94.16%, respectively, and there was no significant difference between these two doses. The 1% dose of the bioformulation was chosen for the other applications to be more economical. In the second stage, whether the bioformulation in question had a negative effect on seedling development was studied. In these tests, the bioformulation was applied to seeds of cultivar susceptible to the pathogen while they were slightly moist (1 g / 100 g seed), after ensuring a good mixture, they were dried for 15 minutes in a clean environment and then placed in petri dishes containing moist filter papers (13 petri dishes, 8 seeds per dish). Seeds without any treatment were considered as control. The bioformulation of this invention did not have any negative effect on germination and seedling development when applied to the seeds of the edible-seeded pumpkin (Table 1). Furthermore, root length, shoot length, seedling vigor index, fresh weight and dry weight increased by 1.20, 1.16, 1.18, 1.39 and 1.07 times, respectively, compared to control (seedlings growing from untreated seed) seedlings.

[0037] Table 1. Germination rates of seeds coated with Trichoderma harzianum (TRIC8) bioformulation compared to untreated seeds and development of the obtained seedlings.

[0038] Root Shoot Seedling Fresh Dry Germination length length vigor weight weight Treatment (43) (co) W Index (ma) (mg)

[0039]

[0040] bioform illation

[0041] (Invention) 87.50 10.04 413 1236.74 57552 9728 In the third stage, the ability of the inventive bioformulation to inhibit disease severity and promote plant growth in the presence of the pathogen F. proliferatum was studied. In these tests, seeds of the cultivar susceptible to the pathogen were inoculated with the pathogen F. proliferatum, then coated with the inventive bioformulation (1 g / 100 g seeds), dried in a clean environment for 15 minutes and then placed in petri dishes (13 petri dishes, 8 seeds per petri dish) with moist filter papers. During said trials, a conidial suspension of TRIC8 isolate (1 x 107conidia / ml + Tween 20, 0.1%) was also prepared for comparison (non-bioformulated version) and seeds were shaken in this suspension for 6 hours. Only seeds infected with the pathogen were considered as positive control (Control +). The pathogen caused 48.08% disease severity in seedlings. However, disease severity was 11.73% when the inventive bioformulation was applied to pathogen inoculated seeds and 14.04% when only conidia suspension of TRIC8 was applied (Figure 3). While the inventive bioformulation inhibited disease severity by 75.6%, the inhibition rate was lower (70.8%) in the conidia-only suspension application of TRIC8. When plant growth parameters were considered, it was observed that all growth parameters were higher in the bioformulation treatment than in the Control (+) (Table 2). The inventive bioformulation increased germination, root length, shoot length, seedling vigor index, seedling dry and fresh weight (2.07, 1.37, 1.38, 2.85, 1.60, 1.16-fold, respectively) even in the presence of the pathogen.

[0042] Table 2. Germination rates of seeds coated with Trichoderma harzianum (TRIC8) bioformulation after Fusarium proliferatum inoculation and development of the seedlings obtained.

[0043] > <

[0044]

[0045]

[0046] In the fourth stage, seeds of the cultivar susceptible to the pathogen were treated with the inventive bioformulation (1 g / 100 g seeds) and sown in soil contaminated with the pathogen (Fusarium proliferatum) and disease severity and some growth parameters were determined for 30 days. The trials were conducted with 5 replications. During said trials also, a conidial suspension of TRIC8 isolate (1 x 107conidia / ml + Tween 20, 0.1%) was also prepared for comparison (non-bioformulated version) and seeds were shaken in this suspension for 6 hours. At the end of this period, the inventive bioformulation was 86.5% effective in reducing disease severity (Table 3). It also increased the number of leaves (1.2-fold), shoot length (1.23-fold), shoot fresh weight (1.56-fold) and shoot dry weight (1.35-fold) compared to control (+) (only plants developed from seeds infected with the pathogen). Seed treatment with TRIC8 without bioformulation was less effective (82.7%) on disease severity but increased growth parameters.

[0047] Table 3. Disease severity and some plant growth parameters 30 days after sowing of seeds treated with Trichoderma harzianum (TRIC8) bioformulation in soil contaminated with pathogen (Fusarium proliferatum).

[0048]

[0049] Industrial Applicability of the Invention

[0050] The invention relates to a granular bioformulation containing Trichoderma harzianum (NCBI accession number: MH351669) isolate TRIC8, supplemented with wheat straw and sugar beet molasses, developed to prevent root and crown rot disease caused by seed-borne Fusarium proliferatum in edible-seeded pumpkin used in the production of snacks, confectionery, and desserts, and to promote plant growth, and is industrially applicable.

[0051] The invention is not limited to the above descriptions and the person skilled in the art can readily present other different embodiments of the invention. These should be considered within the protection scope of the invention claimed by the claims. REFERENCES

[0052] [1] Snowdon, A.L. (2010). A colour atlas of post-harvest diseases and disorders of fruits and vegetables, Vol: 2, Vegetables, Manson Publishing, United Kingdom, 12-51 p.

[0053] [2] Kurt, §. 2012. Bitki fungal hastaliklan. Akademisyen Kitabevi Yaym Dagitim ve Pazarlama Ltd. §ti. Ankara, 214s

[0054] [3] Stoner, M.F. 1981. Ecology of Fusarium in noncultivated soils. (P.E. Nelson, T.A. Toussoun and R.J. COOK, eds.) Fusarium diseases, biology, and taxonomy. The Pennsylvania State University Pres, University Park., 276-286p.

[0055] [4] Katan, J. 1996. Soil solarization: Integrated control aspects. In: Hall, R. [Ed.] Principles and Practice of Managing Soilborne Plant Pathogens. APS Press, St. Paul, MN, USA. pp. 250-278.

[0056] [5] Meyer, J.M., Azelvandre, P., Georges, C. 1992. Iron metabolism in Pseudomonas: salicylic acid, a siderophore of Pseudomonas fluorescens CHAO. Biofactors 4: 23-27.

[0057] [6] Sneh, B., Dupler, M., Elad, Y., Baker, R. 1984. Clamidospore germination of Fusarium oxysporum f.sp. cucumerinum as affected by fluorescent and lytic bacteria from fusarium-suppresive soil. Phytopathology, 74: 1115-1124.

[0058] [7] Janisiewicz, W., Korsten, L. 2002. Biological control of post-harvest diseases of fruits. Annual Review of Phytopathology, 40: 411-441.

[0059] [8] Fravel, D.R. 2000. Commercial biocontrol products, available for use against plant pathogens, http: / / www.oardc.ohio-state.edu

Claims

CLAIMS1 . A bioformulation for the prevention of root rot disease caused by seed-borne Fusarium proliferatum and for the promotion of plant growth in edible-seeded pumpkin, characterized in that it comprises Trichoderma harzianum (NCBI accession no: MH351669) TRIC8 isolate, wheat straw and sugar beet molasses diluted with distilled water.

2. A bioformulation according to claim 1 , characterized in that it comprises 2.0-3.0% Trichoderma harzianum (NCBI accession no: MH351669) TRIC8 isolate, 37.0- 41.0% wheat straw and 57.0-60.0% of the sugar beet molasses diluted with 8- 12% distilled water.

3. A bioformulation according to claim 2, characterized in that it comprises 2.3% Trichoderma harzianum (NCBI accession no: MH351669) TRIC8 isolate, 39.1% wheat straw and 58.6% of the sugar beet molasses diluted with 10% distilled water.

4. The preparation method of a bioformulation for the prevention of root rot disease caused by seed-borne Fusarium proliferatum and for the promotion of plant growth in edible-seeded pumpkin, characterized in that it comprises the processing steps of:i. soaking the ground wheat straw with distilled water, then removing the excess water and adding sugar beet molasses diluted with distilled water, ii. sterilizing the resulting mixture in autoclave and adding Trichoderma harzianum TRIC8 isolate from a conidial suspension,iii. drying in a sterile environment by mixing every day,iv. after the drying process, passing it through sterilized sieves in a sterile environment, taking into sterile jars, and closing the lids with vacuum.

5. The method according to claim 4, characterized in that it comprises the processing steps of:i. soaking ground wheat straw in distilled water at 250-350 ml / 100 g of straw for 1.5-2.5 hours, removing the excess water, adding sugar beet molasses, diluted to 8-12% with distilled water, at 140-160 ml / 100 g of straw,ii. sterilizing the mixture obtained by autoclaving it 3 times at 121 °C for 20 minutes at 1-day intervals and adding 5-7 ml / 100 g of straw from the conidial suspension of Trichoderma harzianum TRIC8 isolate (1 x 107conidia / ml).iii. drying in a sterile environment at 24-26°C for 4-6 days by stirring daily, iv. after the drying process, passing it through sterilized sieves with a mesh size of 0.5-1.4 mm in a sterile environment, transferring it into sterile jars, and closing the lids with vacuum.

6. The method according to claim 5, characterized in that it comprises the processing steps of:i. soaking ground wheat straw in distilled water at 300 ml / 100 g of straw for 2 hours, removing the excess water, adding sugar beet molasses, diluted to 10% with distilled water, at 150 ml / 100 g of straw,ii. sterilizing the mixture obtained by autoclaving it 3 times at 121 °C for 20 minutes at 1 -day intervals and adding 6 ml / 100 g of straw from the conidial suspension of Trichoderma harzianum TRIC8 isolate (1 x 107conidia / ml). iii. drying in a sterile environment at 25°C for 5 days by stirring daily, iv. after the drying process, passing it through sterilized sieves with a mesh size of 1 mm in a sterile environment, transferring it into sterile jars, and closing the lids with vacuum.

7. The bioformulation prepared by a method according to any one of claims 4-6.

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