Production technologies and application forms of biological products based on Trichoderma fungi

y |) 0 [in TYPES OF THE GENUS Trichoderma (ac

= c to Va, 1969): a — T. Koningii; 6 — T. 02o 0 «\ uae 0 m E o 0%

3rd phase. Complete colonization of the host. Trichoderma penetrates the host mycelium, actively growing inside the cells, leading to their death. The parasite’s enzymes (chitinases, cellulases, glucanases) soften the host cell wall, facilitating the process of further colonization.

All biological products based on fungi of the genus Trichoderma in our country are called trichodermin, the preparative forms of which differ depending on the composition of nutrient media, method of cultivation, carriers, and the titer of the finished product.

Previously, the List of pesticides and agrochemicals included the grain-based product Trichodermin-BL. It became widespread due to the ease of production in biolaboratories. The nutrient substrate for mass production of the dry product by the solid-phase method is grain or its waste, less often straw cuttings, beet pulp, sunflower husks, peat, etc. The sterile substrate is inoculated with a starter culture and cultivated at 24-25° for several days (different strains have different growth durations). Then, the biomass is poured into open containers in a layer no more than 3 cm thick until abundant dark green sporulation forms. The mature biomass is dried at 30-35° with good ventilation and periodic stirring. The moisture content of the finished product should not exceed 8%. The product is packed in paper bags. For sanitary and hygienic reasons, the production of the grain-based product is being reduced, although it is still used in a number of laboratories.

The spore-mycelial mass of the fungus with a titer of at least 10 billion spores/g is produced by a number of domestic firms and many regional biological factories of STAZR. The most concentrated dry spore-mycelial product (up to 100 billion spores per 1 g) is obtained by cultivating the fungus on a liquid nutrient medium, followed by drying and grinding the biomass. In practice, the concentrated product is used relatively rarely due to its high cost. The main preparative forms of trichodermin are a dry product on a carrier (grain-based) and a liquid (wet) one. The liquid form has its advantages: the product does not dust, there are no drying costs, and it is easy to prepare working solution for application. However, such a product can be stored for no more than two weeks at a temperature not exceeding 12°. Its activity depends on the viability of conidia developing during the cultivation process. As a rule, the effectiveness of such products decreases sharply with an increase in storage time, which affects the scale of trichodermin application. Trichodermin is also produced by the submerged fermentation method. The fungus biomass is grown in a liquid nutrient medium in continuous cultivation apparatuses (fermenters) or in three-liter jars on shakers. For this purpose, the All-Russian Research Institute of Agricultural Machinery produces two-tier shakers with a capacity of 49, 72, 97, or 128 jars. The cultivation cycle (after inoculation with the starter culture) is 2-3 days.

Fungi of the genus Trichoderma synthesize growth stimulants, antibiotic substances, and lytic enzymes (cellulases, chitinases, proteases). Fresh trichoderma mycelium is twice as active against phytopathogens as surface conidia. However, the shelf life of raw mycelium at +20° is two weeks, surface conidia in dry form — 1.5 years, and dry chlamydospores — 2.5-3 years.

Spectrum of action. Trichodermin is used primarily to suppress the development of soil-borne phytopathogenic fungi of the genera Rhizoctonia, Pythium, Fusarium, Phytophthora, Alternaria, Botrytis, and some other pathogens. Individual strains of trichoderma colonize the surfaces of roots and leaves. The protective properties of these fungi are also manifested in their ability to induce systemic acquired resistance and stimulate plant growth.

— kenia Fig. 203. ANTAGONISTIC RELATIONSHIPS BETWEEN Trichoderma AND Fusarium ON AGAR MEDIUM

Biology. The fungus possesses a wide spectrum of antagonistic properties — hyperparasitism, competition for nutrient substrate, and it produces antibiotics and other substances that inhibit the vital activity of phytopathogens. It produces the antibiotics viridin, gliotoxin, etc.

Application. Soil is the main habitat of fungi of the genus Trichoderma, and it is here that the entire complex of their antagonistic properties is manifested. Therefore, Trichodermin is used mainly against soil pathogens (root and stem rots, fusariosis, damping-off, white and gray rots, etc.). In recent years, reports have appeared on the high effectiveness of trichodermin on strawberries against gray rot. High effectiveness (at the level of 72-78%) of strains 119/80 and SK-14-85 in the fight against late blight of tomatoes has been noted. In addition, trichodermin has a phytostimulating effect. The product is compatible with mineral top dressing and some pesticides. The bulk of trichodermin is applied after soil disinfection by steaming or fumigation. Application rates depend on the species and strain of the fungus, titer, and other conditions, which is why it is necessary to be guided by the application instructions for a specific batch of the product or the List of pesticides... 2001. Usually, the product is applied so that a dose of 10 billion spores is delivered per plant.

'Trichodermin is also applied by spraying a spore suspension. Experiments have shown high efficacy of Trichodermin application on tomato and strawberry for the control of Ascochyta blight, gray mold, and white mold.

For the stem form of cucumber white mold, a method of applying the antagonist fungus in the form of a paste — stem coating — has been developed. This also reduces the intensity of the development of Ascochyta blight, Phoma blight, and Alternaria blight. Trichodermin-BL (currently not included in the List... 2001. Active ingredient: Antagonist fungus

Biological products based on fungi of the genus Trichoderma

Biological products based on fungi of the genus Trichoderma are an effective tool for plant protection of vegetable and ornamental crops against root rots and wilting. They act as direct antagonists of pathogens, but their efficacy strongly depends on the conditions of application and the composition of the soil microflora. In practice, two forms of the product are most often used: dry powder and liquid. The dry powder based on spore-mycelial mass of Trichoderma lignorum (in the original text — Тиисподеттла Вопотлит) contains spores, mycelium, and residues of the nutrient substrate. The liquid form contains mycelium and spores of Trichoderma harzianum (in the original text — ТГисводетта Нопогит), as well as the antibiotics viridin and gliotoxin.

The main target objects for the application of these forms are cucumber root rots and carnation Fusarium wilt. To control root rots of cucumber, plants are irrigated with a 0.5% suspension of the product at one-month intervals. Against Fusarium wilt of carnation, planted cuttings are sprayed with a 0.4% working solution also once a month. The granular form of the product (Trichodermin G) contains the TVD-93 strain of the fungus Trichoderma harzianum (in the original text — Тисбодента Нопонмит). It is used against cucumber root rots, tomato and cucumber gray and white stem rots, as well as strawberry gray mold.

• Titer of dry powder — from 10 billion spores/g
• Titer of Trichodermin G — 2 billion spores/g
• Fungus activity depth — up to 10 cm
• Irrigation rate for cucumber — 100–200 ml/plant

Hyperparasitic products based on Ampelomyces and Coniothyrium

Ampelomycin and Coniothyrin are biological products based on hyperparasitic fungi that destroy disease pathogens directly on the affected plants. The active ingredient of Ampelomycin is the pycnidial fungus Ampelomyces quisqualis (order Sphaeropsidales, class Deuteromycetes). Under natural conditions, this hyperparasite actively colonizes and destroys the mycelium, conidia, and cleistothecia of powdery mildew fungi of the genera Erysiphe and Sphaerotheca. The product effectively protects cucumber, tomato, grape, and ornamental crops.

In practice, several formulation forms of Ampelomycin are used. The basic version is a dry spore-pycnidial-mycelial mixture on barley residues with a working suspension titer of 2 × 10⁹ spores/ml (in the source — 2 × 10°). A liquid form of the product with a higher titer has also been developed in one of the regions. For the protection of grapes, pumpkins, strawberries, tomatoes, and ornamental plants, water-dispersible granules AQ10 based on hyperparasite pycnidia are produced abroad.

The biology of the hyperparasite's interaction with the host has its own features. Conidia of Ampelomyces quisqualis germinate into a germ tube, which pierces the membrane and penetrates into the cells of powdery mildew. The infected cell swells, and within 3–5 days, darkening pycnidia form inside it, staining the white disease coating gray. Pycnospores are washed off by raindrops, dispersed by wind or insects, and in the presence of surface moisture, cause new infections. The entire development cycle on the host takes only 5–6 days, and the first signs of infection are visible as early as the 3–4th day.

1. Monitoring: carry out an inspection and identify the first signs of powdery mildew on the plants.
2. Solution preparation: dilute the preparation immediately before use.
3. Treatment: spray the plants in cloudy weather or in the evening at a temperature of 24–26 °C and air humidity from 85%.
4. Re-application: carry out a second treatment after 8–9 days to consolidate the result.

• Working titer of the suspension — 2 × 10⁹ spores/ml
• Temperature for treatment — 24–26 °C
• Air humidity — from 85%
• Interval between treatments — 8–9 days
• Development period on the host — 5–6 days

Coniothyrin is another hyperparasite preparation, not yet included in the official List of pesticides. Its active ingredient is the pycnidial fungus Coniothyrium minitans (order Sphaeropsidales, class Deuteromycetes). The preparation is produced in the form of a mixture of conidia and pycnidia of the fungus Coniothyrium minitans. Its production and application technology is similar to the technology of manufacturing Ampelomycin.

Gliocladium: application features and dosages

Gliocladium is a biological preparation based on the antagonist fungus Gliocladium virens (class Deuteromycetes). In nature, this hyperparasite inhabits the soil, on plant residues, as well as on sclerotia and microsclerotia of pathogens (Sclerotinia, Sclerotium, Botrytis, etc.). The biological agent significantly reduces the infection load in the soil, improves plant nutrition, and stimulates their growth. By its mechanism of action, it is similar to Trichodermin.

The preparation is produced in the form of a grain substrate or a spore-mycelial culture liquid. It is applied to the soil in the form of an aqueous suspension or dry powder, and is also used for seed treatment. Gliocladium effectively suppresses white rot of cucumber and tomato, and in open ground, it protects plantings of sunflower and carrot.

For the control of root rots of vegetable crops in open ground, dry powder is applied to the soil at a dose of 40 kg/ha. Soil drenching with an aqueous suspension with an application rate of 100 l/ha is also practiced. The treatment allows reducing the incidence of root rot, brown spot, and ascochyta blight in plants. In closed ground, this form of the preparation has not yet found application and is undergoing testing.

Paecilomyces and Mycostop: protection against nematodes and root-zone infections

The fungus Paecilomyces lilacinus is a typical inhabitant of soils and the plant rhizosphere. This species is capable of parasitizing on eggs and females of root-knot nematodes (genus Meloidogyne), as well as on cyst-forming and other sedentary nematodes. The infection process begins with the penetration of hyphae into egg sacs. The hyphae entwine the eggs from the outside, penetrate inside, and absorb their contents, with young eggs being the most susceptible to infection. From the egg sacs, hyphae can penetrate the female's body through the vulvar opening.

The biological preparation Biocon based on this fungus shows high effectiveness against phytoparasitic nematodes, not inferior to chemical nematicides. The fungus possesses high physiological plasticity, which allows it to develop in various types of soils. In closed ground, it is recommended to apply the preparation according to the following sequential scheme.

1. Soil steaming.
2. Thorough drenching of the soil cooled to 25–32 °C with a liquid culture of the fungus at a dose of 25–50 ml per 4–6 l of water per 1 m² (the rate is specified for an average titer of 5 billion conidia per 1 ml).
3. Mixing the soil with a tiller to a depth of 30 cm.
4. Abundant wetting of the planting holes with water on the eve of the second treatment.
5. Application of the deep culture of the fungus into the planting holes a few days before planting the transplants at a dose of 6–8 ml in 250–350 ml of water per one planting hole.
6. Dipping the roots of transplants into the conidia suspension immediately before planting (dilution with water in a ratio of 1:25–1:30).
7. Irrigation of beds with the fungus culture at a dose of 15–30 ml per 2.5–3.5 l of water per 1 m². The treatment is carried out 1–3 times during the growing season at an interval of 25–35 days as needed upon detection of meloidogyne infestation foci.

• Reduction in nematode infestation — by 60–97%
• Total conidia consumption — 500–800 billion/m²
• Root infection in cucumbers and tomatoes — 1–2 points
• Root infection in control — 3–4 points

For disease control, the biological preparation Mikostop, SP is also used. Its active ingredient consists of spores and mycelium of the strain Streptomyces griseoviridis. The preparation is produced as a wettable powder with a titer of at least 10*–10? cells/g. It has low toxicity for warm-blooded animals, and manual work in treated areas can be resumed just one day after application.

Protection technology for carnations and the potential of natural antibiotics

To combat fusarium wilt in carnations, a combined treatment scheme for planting material and substrate is used. The method combines pre-planting cutting soaking and regular soil irrigation. This makes it possible to block the development of the pathogen in the early stages of plant growth and protect the root system during the active growth period.

1. Before planting, immerse the cuttings for 15 minutes in a 0.01% working solution of the preparation. The liquid consumption is 10 L per 3,000 cuttings.
2. One day before planting the cuttings, carry out the first spraying of the soil with a 0.05% solution.
3. One month after planting, carry out a second spraying of the soil with a 0.05% solution.
4. Conduct subsequent preventive soil sprayings during the growing season at one-month intervals. The working liquid consumption for all soil treatments is 1 L per 10 m².

Natural antibiotics — biologically active substances produced by microorganisms — are becoming an alternative to chemical pesticides in professional crop production. They selectively suppress or destroy viruses, bacteria, actinomycetes, fungi, algae, and protozoa. Unlike organic acids or alcohols, antibiotics work in ultra-low concentrations. For example, penicillin has a bactericidal effect on sensitive bacteria at a dose of only 0.000001 g/mL. At the same time, they possess high selectivity, which distinguishes them from general biological poisons such as cyanides, corrosive sublimate, or arsenic.

The biological activity of antibiotics is measured in conventional action units per 1 mL of solution (units/mL) or 1 mg of dry preparation (units/mg). One unit is defined as the minimum amount of substance capable of stopping the growth of a test strain in a specific volume of medium. One unit of penicillin activity inhibits the development of the 209 strain of Staphylococcus aureus in 50 mL of broth, and streptomycin — of E. coli in 1 mL. After the chemical synthesis of many antibiotics, it became possible to convert units of action into mass: thus, 1 mg of pure streptomycin base is equivalent to 1000 units of biological activity.

Systemic antibiotics: global experience and regulations for phytobacteriomycin

In crop production, more than 3,000 described antibiotics are used, classified by the origin of producers, mechanism of action, chemical structure, and spectrum of activity. For field application, the systemic properties of the preparation are important. Components must easily penetrate tissues and move through the vascular system. This allows not only for the suppression of pathogens but also for the neutralization of their toxins and enzymes, while simultaneously stimulating the immunity and growth of crops.

Global practice confirms the high profitability of such treatments. In Japan, since 1961, the actinomycete antibiotic blasticidin-S has been used against rice blast. Since 1965, the systemic antibiotic kasugamycin (kasumin) has been on the market with an annual production volume of 20,000 tons. It protects beans, peppers, eggplants, sugar beet, apple trees, and pear trees from 8 species of phytopathogenic fungi by penetrating the tissues and blocking the germination of pathogen spores.

In domestic practice, the standard for bactericidal protection is phytobacteriomycin (FBM) — an antibiotic of the streptothricin series. The preparation is produced as a yellow-gray powder (the pure substance is a water-soluble amorphous cream-colored powder) with an activity of 300,000 units per 1 g. FBM is highly effective against the causative agents of root, mucous, and vascular bacteriosis, as well as against some fungal pathogens, including rhizoctonia.

• Retention of activity in tissues — from 9 to 38 days
• Biological activity of the preparation — 300,000 units/g
• Storage temperature in the warehouse — from -20 °C to +40 °C
• Guaranteed shelf life — 1 year

Phytobacteriomycin is rapidly distributed through the plant's vascular system. At recommended application rates, it exhibits no phytotoxicity and provides a pronounced stimulatory effect on plant growth and development. The product is completely safe for beneficial entomophages and pollinating bees, allowing it to be used successfully in integrated plant protection systems.

When using antibiotics in plant protection, it is important for the agronomist to remember two main risks: pathogens adapt rapidly to active ingredients, and the preparations themselves can cause allergies in personnel. Nevertheless, proven solutions exist for controlling bacterial diseases, such as Phitolavin-300 (manufacturer — "Pharmbiomed", hazard class III). The preparation is effective on tomatoes against bacterial canker, pith necrosis of the stem, Fusarium wilt, and root rots. On cabbage, it is used for protection against bacterial diseases and black leg.

• Hazard class of the preparation — III
• Concentration for seed treatment — 0.2%
• Seed soaking time — 2 hours
• Interval between transplant treatments — 15 days

Trichothecin: specifics of application and risks of phytotoxicity

The preparation Trichothecin, WP (10% wettable powder) contains the antibiotic of the same name, produced by the Trichothecium roseum culture. It is effective against many fungal pathogens, especially against powdery mildew on cucumber. However, the preparation is moderately toxic to warm-blooded animals, irritates mucous membranes and skin, and is also a mild allergen. Due to frequent cases of plant burns and allergic reactions in humans, this preparation was previously produced in small quantities and was not included in the List of Pesticides and Agrochemicals.

1. One day before the planned treatment, set aside several test plants in the greenhouse.
2. For transplants, prepare test solutions with concentrations of 0.04%, 0.08%, and 0.10%.
3. For fruiting plants, use test concentrations of 0.05%, 0.1%, and 0.2%.
4. Carry out spraying of the test plants and evaluate their condition for signs of burns.
5. In the absence of signs of phytotoxicity, treat the plantings at the first symptoms of the disease with a working solution concentration of 0.04–0.2% and an application rate of the preparation of up to 2 kg/ha.