RU2634415C1 - Fungus strain trichoderma asperellum for obtaining biological preparation of complex action for crop growing - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of the microscopic fungus Trichoderma asperellum OPF-19, which has antagonistic activity in relation to phytopathogenic microorganisms, was deposited in the Russian National Collection of Industrial Microorganisms under the registration number of VKPM F-1323 and can be used to create a complex biological preparation for use in crop growing, including the conduct of organic farming.

EFFECT: it allows to increase the yield of agricultural plants.

4 dwg, 13 tbl, 9 ex

Description

The invention relates to agricultural microbiology and biotechnology, relates to biological agents for protecting plants from phytopathogenic microorganisms and stimulating their growth, as well as for improving biodegradation of crop residues, and is a strain of the microscopic fungus Trichoderma asperellum OPF-19.

Currently, in the development of biological products for crop production, researchers give preference to those strains of microorganisms that have a pronounced integrated biological activity. Many types of microorganisms have revealed multiple ways to improve plant life, for example, production of stimulating phytohormones, optimization of mineral nutrition by fixing molecular nitrogen or increasing the availability of sparingly soluble compounds of macro- and microelements, inducing systemic resistance of plants to adverse biotic and abiotic environmental factors, direct suppression of the development of phytopathogens and phytophages, biodegradation of toxic compounds, etc. Among bacteria, the representatives of the Bacillus and Pseudomonas genera possess the greatest biotechnological potential as the basis for biological products for plant growing; among fungi - representatives of the genus Trichoderma. Among the advantages of the latter, which in some cases can play a decisive role in choosing a biological product based on a particular microorganism, the following should be noted: the ability to form conidia, which can remain viable for a long time under adverse environmental conditions; high growth rate; low inertia, which manifests itself in the rapid activation of spores and the "explosive" nature of growth on rich substrates; low demands on environmental conditions; a tendency to acidic pH values and, accordingly, the ability to exhibit its biological activity in acidic soils; the ability to hyperparasitism on phytopathogenic fungi, which together with the production of antibiotic substances can effectively protect plants. However, the practical potential of representatives of this kind is revealed, in our opinion, is not enough.

Trichoderma viride Pers. ex. SF Gray under the numbers F-3271D (RF patent No.2013054, publ. 05/30/1994), F-3269D (RF patent No. 050528, publ. 04/15/1994), F-3273D (RF patent No. 20133055, publ. 30.05.1994 ), as well as Trichoderma harzianum Rifai under the numbers F-3270D (RF patent No.2010527, publ. 04/15/1994) and F-3272D (RF patent No. 20150526, publ. 04/15/1994) to obtain preparations against causative agents of gray rot of grapes, in primarily to combat Botrytis cinerea, as well as some species of Penicillium spp., Aureobasidium pullulans. These strains can reduce rot losses during storage and increase the yield of grapes suitable for storage, as well as reduce the number of chemical treatments.

Known strain Trichoderma sp. MG-97 (RF patent No. 2171580, publ. 08/10/2001), used to protect coniferous seedlings from fusarium infections. The strain exhibits pronounced antagonistic activity against Fusarium avenacem, F. sporotrichiella v. poae, F. moniliforme v. lactis, F. nivale, F. gibbosum.

The disadvantage of these inventions is the lack of biological activity, manifested only in the protection of certain plant species from only certain phytopathogens. However, their effect on plant growth and yield remains unknown.

A known method of protecting plants of cucumber and tomato from phytopathogens (RF patent No. 2094991, publ. 10.11.1997), involving the treatment of affected plants with a paste-like preparation based on the Trichoderma harzianum VKM F-2477D strain with the addition of carboxymethyl cellulose. This ensures the complete recovery of stems of cucumber and tomato (with a weak degree of damage of 0.1-1 points), affected by ascochitosis, white and black rot, and increase productivity.

Known strain Trichoderma lignorum NII KKM SSC BB "Vector" F5 (RF patent No. 2121793, publ. 11/20/1998), which has antagonistic activity against phytopathogenic fungi Fusarium sp., Botrytis cinerea and bacteria Pseudomonas corrugata, which reduces the incidence of serum morbidity and morbidity. rotting and hollowness caused by these pathogens, and increasing productivity. When a liquid preparation based on the strain is introduced into the soil, the phytopathogenic potential decreases in the root zone of plants.

The disadvantages of these strains include the limited scope of their use in closed ground and two cultures, as well as a narrow range of diseases in the fight against which the strains were effective.

Known strain Trichoderma viride 23, with fungicidal properties (RF patent No. 2186847, publ. 08/10/2002). The strain exhibits antagonistic activity against Botrytis cinerea, Fusarium lini, Fusarium solani, Fusarium oxysporum, Penicillium verrucosum, Pythium debaryanum, protects plants from diseases in the field and contributes to increased productivity. The disadvantage of the strain is a narrow spectrum of suppressed phytopathogens. In addition, nothing is known about the effect of the strain on plant growth.

A known strain of the fungus Gliocladium catenulatum Gilmann et Abbott D-553 (RF patent No. 2307156, publ. 09/27/2007) to obtain a biological product against fungal pathogens of vascular diseases and root rot of plants. The strain is antagonistic to Fusarium solani, F. culmorum, Verticillium dahliae; well takes root and is actively developing in various types of soils, where at the same time there is a decrease in the number of propagules of Verticillium dahliae; stimulates the germination and growth of cucumber seedlings. The disadvantage of this invention is directed only to the fight against phytopathogens, the biological activity of the strain, a narrow range of suppressed phytopathogens. There is practically no information about the effect of the strain on growth indicators and plant productivity.

As a prototype of the claimed invention can be considered a strain of Trichoderma viride Pers. ex. S. F. Gray 16 CCM F-59M 80-90 (RF patent No. 2170511, publ. 07.20.2001). The strain has high hyperparasitic activity against a wide range of plant pathogens belonging to the genera Fusarium, Helminthosporium, Rhizoctonia, Pythium, Sclerotinia, Alternaria, Verticillium, etc., is resistant to a number of chemical insecticides and fungicides; reduces the incidence and increases the yield of plants (for example, two varieties of spring barley). The main disadvantage of the prototype can be considered that the biological activity of the strain is limited to its use as the basis for biofungicide for presowing treatment of seeds. However, it is not known whether this micromycete, after harvesting, is able to carry out biodegradation of stubble and help reduce the infectious background in soils of agrocenoses.

The objective of the claimed invention is the selection of a new natural strain with antagonistic activity against phytopathogenic microorganisms, protecting plants from diseases, stimulating their growth and increasing productivity, resistant to chemical plant protection products and not inhibiting beneficial soil microflora.

This problem is solved thanks to the isolation and use of the Trichoderma asperellum strain OPF-19, deposited in the All-Russian Collection of Industrial Microorganisms under the number F-1323. The strain was isolated from the surface of the roots of tomatoes grown in open ground in the Vysokogorsky district of the Republic of Tatarstan. The species affiliation of the culture was determined on the basis of the results of sequencing of the 16S rRNA gene at the State Scientific Research Institute of Agricultural Microbiology of the Russian Agricultural Academy. The claimed strain has the following characteristics.

Cultural-morphological and physiological-biochemical characteristics

After 72 hours of growth on potato glucose agar (KGA) at 30 ° C, the colony of strain T. asperellum OPF-19 has a diameter of 55-65 mm, at 40 ° C - 2 mm. Colonies grown on the KGA form several concentric rings, on the surface of which intensive sporulation is noticeable. In the central part, the colony is darker, earlier acquires a green color, aerial mycelium forms as it moves away from the center. When cultured at 25 ° C in the absence of light, conidia form after 35-45 hours. Pigment diffusion into agar does not occur; in old colonies there is a faint specific odor.

When growing on Saburo agar medium for 5 days and 25 ° C, pads form abundantly on the entire surface of the colony. Spore-bearing extensions on conidiophores are absent. Sterile processes are not formed. Conidiophores are more often formed in the pads and rarely on the aerial mycelium; conidiophores are symmetrical and terminate in four phialids. Paired branches are formed below the apex of the conidiophore and are located at an angle of about 90 ° with respect to the main axis. The primary branches lengthen as they move away from the apex, paired branches have the same length, non-branching lateral branches of the second order form on the lateral branches of the first order. The width of the conidiophore is 2.1-5.0 microns. Phialids form at the ends of branches of the first and second order, clusters of 2-4 phialids form. The phialides are straight, bulbous, slightly expanded in the middle, length - 5-10 microns, width - 2.2-5.7 microns. Intercalary phialides do not form. Conidia have a dark green color, the shape is a little ovoid, often closer to spherical, with a diameter of 3.5-4 microns.

Chlamydospores are formed by deep cultivation, pale green or brown.

Physiological and biochemical characteristics

The strain is a strict aerobic, saprotroph.

The temperature is optimal for growth at 27 ° C, while the strain can grow in the temperature range from +10 to + 37 ° C. Optimum pH of the medium is in the range of 6.5-7.2, growth is observed at pH values from 3.0 to 8.0.

It utilizes glucose, galactose, fructose, mannose, arabinose, maltose, sucrose, cellobiose, amygdalin, arbutin, esculin, salicin, trehalose, glycogen, gentiobiose, starch, cellulose, fucose as a source of carbon and energy.

As a source of nitrogen, T. asperellum OPF-19 can use nitrates, ammonium salts, organic nitrogen in the form of amino acids and peptides.

The reproduction of a microorganism can be carried out in several ways. In the surface method, the conidia and mycelium of the strain T. asperellum OPF-19 are applied to the surface of the nutrient medium in Petri dishes, microbiological mattresses or in any microbiological container with a large area of the working surface, then the biomass is distributed over the surface of the medium with a Drigalski spatula, and the vessels are incubated in the thermostat at 27 ° C.

With a deep cultivation method, the conidia of the strain germinate and form mycelium in a liquid nutrient medium in Erlenmeyer conical flasks (either in rocking flasks) or in a fermenter. When culturing conidia and mycelium strain in Erlenmeyer flasks, incubator shakers are used. Flasks filled with nutrient medium 1 / 4-1 / 3 of their volume are inoculated with aliquots of the biomass of the strain with a dense nutrient medium, placed on a shaker incubator and pumped at 27 ° C for 3 days. When cultured in a fermenter, the nutrient medium in the culture vessel is inoculated with the daily mycelium of the strain obtained in the flasks as described above. Potato-glucose or potato-sucrose broth is best suited as a nutrient medium for deep cultivation. Ingredients: potatoes - 200 g / l, glucose or sucrose - 20 g / l, tap water - up to 1 l. The potatoes are crushed, boiled in tap water for 30 minutes, filtered, the filtrate volume is adjusted to 1 l with tap water, glucose or sucrose is added.

The strain inhibits the growth and development of many phytopathogenic fungi, such as Ascochyta pisi, Cercospora beticola, Claviceps purpurea, Sclerotinia sclerotiorum, Alternaria alternata, Botrytis cinerea, Fusarium graminearum, Passalora fulva, Verticillum dahliae. This type of biological activity of the strain is realized both due to the production of antifungal metabolites and due to the ability to hyperparasitism.

Conidia of T. asperellum strain OPF-19 remain viable when exposed to chemical fungicides such as Kolosal, Kolosal Pro, Scalpel, Ferazim, Rayek, Benomil 500, Alto Turbo, Stinger, Inshur Perform, as well as the herbicides Laren Pro, Cortes SP, Tornado.

T. asperellum OPF-19 exhibits growth-stimulating activity against plants, one of the mechanisms of its realization is the production of substances with auxin-like activity.

In addition, the inventive strain has the ability to biological destruction of stubble, an important consequence of which is the reduction of the infectious background in the fields, manifested in a decrease in the incidence of crops in the subsequent culture.

The invention is illustrated by the following examples.

Example 1. The study of the antagonistic activity of a strain of Trichoderma asperellum OPF-19

To identify the antagonism of the studied strain to phytopathogenic microorganisms, the agar block method was used. A suspension of spores and phytopathogenic mycelial fragments was sown on a Petri dish with KHA. At the same time, T. asperellum OPF-19 was sown in a separate Petri dish with the same medium. All microorganisms were placed for 3-5 hours in thermostats at a temperature of 27 ° C so that conidia and spores germinated. After this time, agar blocks were cut from a medium on which T. asperellum OPF-19 culture was seeded, and they were placed with a microbiological needle into Petri dishes with phytopathogens so that the side of the block on which T. asperellum culture was located OPF-19, turned out to be directly adjacent to the surface of the medium with a developing culture of phytopathogen. The control was lawns of phytopathogens sown simultaneously with experimental ones, but to which agar blocks with trichoderma were not placed. Control and experimental plates with cultures were placed in a thermostat at 27 ° C. Observations were carried out on days 2-5, depending on the growth rate of the phytopathogen.

The results of determining the antagonistic activity of the strain T. asperellum OPF-19 are presented in the form of photographs in Figure 1.

According to the information presented, the strain T. asperellum OPF-19 is a fairly strong antagonist and has a wide spectrum of activity; all test objects used in the study showed sensitivity to the antagonistic strain, while the growth of phytopathogens Ascochyta pisi, Claviceps purpurea, Sclerotinia sclerotiorum in vitro was completely suppressed.

Example 2. Laboratory tests of the effectiveness of strain T. asperellum OPF-19

The seeds of winter wheat of the Ivina variety were used as an object of study. The seeds were treated semi-dry with a liquid culture of T. asperellum OPF-19 strain with a titer of 1 × 10 ⁸ conidia / ml obtained on a deep cultivation medium. The control seeds were treated with water, and Bactofit, B (based on the Bacillus subtilis bacterium, strain IPM 215) with a flow rate of 3 l / t recommended by the manufacturer was used as a standard. The flow rate of the working solution is 10 l / t. A day after treatment, the seeds were placed in a moist chamber and kept at 25 ° C. The development of the fungus on the seeds was taken into account after 3 days, and the length of the root and sprout was measured 6 days after being placed in a moist chamber. The results are presented in table 1.

According to the data presented, the optimum rate of liquid culture consumption of T. asperellum OPF-19 with a titer of 1 × 10 ⁸ CFU / ml was 0.8 l / t. Higher consumption rates did not lead to a significant increase in growth or a decrease in seedling infestation. The stimulating and fungicidal effect of seed treatment in all cases was significantly higher than in the control. Also, the stimulation of the growth of seedlings and fungicidal activity were manifested in the case of the use of the T. asperellum OPF-19 strain more than when exposed to Bactofit, J.

Example 3. Tests of the effectiveness of the strain T. asperellum OPF-19 in the growing experiment on peas

A vegetation experiment to identify the physiological activity of T. asperellum OPF-19 was carried out on Vatan peas in plastic vessels with a capacity of 1.5 l filled with soil.

Pea seeds were pre-washed in running tap water using a gauze filter, 6-7 times, after which they were placed on sterile filter paper and dried for several hours. After this, pea seeds were treated semi-dry with a conidia suspension of T. asperellum OPF-19, which was prepared as follows. Initially, conidia were washed from the nutrient medium (KGA) with sterile physiological saline and a suspension with a titer of 1 × 10 ⁸ conidia / ml was obtained, which was determined by counting in a Goryaev chamber. Next, from the initial suspension, a working solution was prepared for presowing treatment of plant seeds at the rate of 1 l of suspension per 10 l of working solution; working solution consumption 10 l / t. The control was plants treated with water. Sowing seeds was carried out two days after treatment; seed embedment depth of 5 cm. The soil was moistened with tap water up to 60% of full moisture capacity.

Closed cuvettes with sown seeds were placed in climatic chambers at a temperature of 23 ° C and in daylight conditions - 18 hours, night - 6 hours. With the advent of seedlings, the covers were removed from the ditch. Watering was carried out as the soil dried up with tap water.

Accounting for germination, growth indicators and the degree of disease prevalence was carried out 14 days after sowing, after washing the plants from the soil mass.

It was revealed that the claimed strain significantly suppressed the development of phytopathogens with which seeds were infected. In Figure 2, it is noticeable that in the control plants the main root is thin with sparse lateral roots, the cotyledons are affected by black spots, brown marks of plant damage by phytopathogens are visible on the stems. In contrast, in plants treated with a suspension of T. asperellum OPF-19 conidia, no such manifestations were observed. The root system had a healthier appearance, better developed lateral roots, which indicates a greater absorption capacity of the roots of these plants, which affects the development of the whole plant as a whole.

In more detail, the data on the effect of T. asperellum OPF-19 on the growth of pea plants are presented in table 2.

Thus, T. asperellum OPF-19 contributes to a significant increase in pea germination and better growth of its vegetative organs, including by suppressing the development of seminal infection.

Example 4. Tests of the effectiveness of the strain T. asperellum OPF-19 in the growing experiment on soybean variety Duet

The experiment, the preparation of a suspension of conidia T. asperellum OPF-19 were carried out in the same way as described in example 3. As a standard, the experiment included the drug Fitosporin M, Zh (LLC NPP Bashinkom), the seed treatment of which was carried out in accordance with the recommendations manufacturer. The results are presented in table 3.

Duet cultivar soybean plants generally proved to be less susceptible to biological treatment. But if in the case of using Fitosporin M seeds for pre-sowing treatment, their growth indices did not differ from the control, then when using a suspension of T. asperellum OPF-19 conidia, plant growth was enhanced: the root length exceeded the control by 15%, shoots by 7%, crude mass - by 10%.

Example 5. Detection of auxin-like activity of strain T. asperellum OPF-19

The biotest is based on the stimulation by auxins of the stretching of coleoptiles of cereals, in particular wheat. Wheat seeds were soaked for 18 hours in tap water, after which they were planted in cuvettes and placed in the dark in a thermostat at 25 ° C for 2 days. For biotest, seedlings with a coleoptile length of 18-20 mm were selected. Coleoptiles were separated, the first sheet was removed and placed on the "guillotine" of 10 pieces so that the cut-off zone (5 mm) was 4 mm below the apex, after which the necessary sections were cut with a sharp blade. The resulting hollow cylinders were temporarily placed in a Petri dish with distilled water (not more than 3 hours). After that, 10 segments of coleoptiles were placed in Petri dishes with a diameter of 3 cm with 3 ml of the studied solutions. Distilled water was used as a control, and a solution of indolyl-3-acetic acid (IAA) at a concentration of 1 mg / L was used as a positive control. Petri dishes were placed in the dark in a thermostat at 25 ° C for 18-20 hours, after which the length of coleoptiles was measured. In the case of the presence of auxin-like activity in the studied solutions, the length of the experimental coleoptiles exceeded the length of the coleoptiles in the control due to the stimulation of cell growth by stretching.

Experimental solutions were prepared by diluting a liquid culture of T. asperellum OPF-19 strain with a titer of 1 × 10 ⁸ conidia / ml, obtained on a deep cultivation medium, 10 times and 100 times. The results are presented in table 4.

Thus, the strain T. asperellum OPF-19 produces metabolites (possibly auxins, but not necessarily) that stimulate cell growth by stretching, as well as IAA, i.e. it can be concluded that the strain exhibits auxin-like activity.

Example 6. Evaluation of the effectiveness of processing plants with a strain of T. asperellum OPF-19 in the field

For the experiment, a liquid culture of T. asperellum OPF-19, grown on a medium for deep cultivation, with a titer of 1 × 10 ⁸ CFU / ml was used. The assessment was carried out in field small-plot experiments on spring wheat plants of the Ekada 66 variety in 2014 and 2015. In 2014, pre-sowing treatment with a conidial suspension of trichoderma in various dosages and spraying of vegetative plants with a consumption rate of 2 l / ha were used as treatment methods. In 2015, pre-sowing treatment was carried out at the rate of 1 l of conidial suspension per 1 ton of grain, spraying by vegetation - 2 l / ha. The results are shown in tables 5-10.

The experimental data indicate a reliable biological effectiveness of the strain T. asperellum OPF-19, expressed in a decrease in the incidence and increase the yield of spring wheat.

Example 7. Evaluation of the compatibility of the strain T. asperellum OPF-19 with chemical plant protection products

The experiment was carried out as follows.

Prepared working solutions of pesticides according to the instructions of the manufacturers, to which was added a liquid culture of strain T. asperellum OPF-19. In a control, the same strain culture was added to sterile tap water. Assessment of the viability of the strain was carried out after 3 hours and 24 hours after the introduction of the culture of the fungus in the working solutions of pesticides. The results are presented in table 11.

According to the data presented, in working solutions of the studied pesticides, the strain remains viable no worse than in water even after 24 hours of incubation, which suggests its compatibility with many chemical plant protection products.

Example 8. The study of the ability of the strain T. asperellum OPF-19 to carry out the destruction of crop residues in the laboratory

The experiment evaluated the loss of mass of straw under the influence of T. asperellum OPF-19. Straw was harvested in early September 2015 from the field after harvesting spring wheat. In the laboratory, it was dried to an air-dry state, after which sections of former wheat stalks (leaf parts from the experiment were deliberately excluded) were selected from a randomly taken beam, which had little or no damage to other microorganisms in appearance. 6 cm long segments were obtained from them, which were individually weighed and individually sterilized by autoclaving at 1.1 atm, after which 2 sterile tweezers were placed in Petri dishes on an agar medium, which was used as a tap water with 0.1% lactose, which, according to the literature, is a stimulant of cellulolytic activity. After this, a culture of strain T. asperellum OPF-19 was sown perpendicular to the medium. The plates were incubated for 18 days in the dark at room temperature (no higher than 22 ° C). After this time, a visual assessment was made of the strain growth on the medium and straw sections, as well as washing and drying of straw sections to an air-dry state in order to determine their mass. The results are presented in table 12.

According to the results, the strain T. asperellum OPF-19 showed high efficiency. The strain culture developed well on sections of straw, which testifies to the ability of the fungus to use plant residues as a source of nutrients. The strain also demonstrated significant ability to reduce the mass of straw in vitro; Given the short duration of the experiment, it can be assumed that with a longer exposure to T. asperellum OPF-19 is able to effectively destroy difficult to decompose plant residues.

Example 9. Evaluation of the aftereffect of stubble treatment with T. asperellum OPF-19 strain in a subsequent culture

The experiment was carried out in the area where the stubble remained after harvesting winter wheat. The site was divided into strips with an area of 30 m ² , in each lane one variant of the stubble processing experience was laid: 1) control 1 (without treatment); 2) control 2 (solution of ammonium nitrate 10 kg / ha); 3) Sternifag preparation, joint venture (LLC AgroBioTehnologiya, Russia), 80 g / ha - as a standard; 4) liquid culture of T. asperellum OPF-19 - 0.3 l / ha; 5) liquid culture of T. asperellum OPF-19 - 0.8 l / ha; 6) liquid culture of T. asperellum OPF-19 - 1.5 l / ha. In all variants (except control 1), ammonium nitrate at the rate of 10 kg / ha was added to the working solutions for stubble treatment prepared at a rate of 200 l / ha. After treatment with the preparations, the crop residues were planted into the soil no later than 4 hours later. After treatment, the whole plot was sown with winter wheat of the Kalym variety.

In the spring, after wintering, when the wheat reached the “tillering end” phase of development, plants were selected for accounting. It was found that plants are affected by cercosporellosis and rhizoctonia root rot. The accounting data are shown in table 13.

Significant differences in the coefficient of tillering of plants, the prevalence of root rot relative to both controls were in the variant with T. asperrellum OPF-19 - 1.5 l / ha. As for the development of the disease, this option was significantly different not only from both controls, but also from other options, including the standard.

Thus, the technical result of the claimed invention is the reduction of infection by phytopathogens of vegetative plants and crop residues, stimulation of growth of agricultural plants, resulting in an increase in their productivity. It is achieved by the fact that Trichoderma asperrellum OPF-19 strain of micromycetes is used as a plant treatment agent, which has a range of useful properties, such as the ability to inhibit the growth of phytopathogenic fungi and protect plants from diseases, stimulate the growth of seedlings, including due to auxin-like activity , increase productivity, carry out biological destruction of crop residues and reduce the infectious background in the soil, and at the same time resistant to chemical pesticides.

Claims (1)

A strain of the microscopic fungus Trichoderma asperellum, deposited in the All-Russian Collection of Industrial Microorganisms under the number F-1323, to obtain a biologic preparation of complex action to protect agricultural plants from diseases caused by phytopathogenic fungi, stimulate their growth and increase productivity, as well as biodegradation of crop residues and improve phytosanitary condition soil compatible with chemical pesticides.

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