RU2750957C2 - Trichoderma viride strain - producer of soyasaponin with antifungal and growth-stimulating activity against plants and earthworms - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. The Trichoderma viride strain, a producer of soyasaponin I, with high antifungal and growth-stimulating activity, was deposited in the All-Russian Collection of Industrial Microorganisms under the number F-1525.

EFFECT: provides suppression of the development of root infections, stimulates seed germination and plant growth, increases the productivity of Eisenia fetida vermiculture, used for processing organic residues.

1 cl, 2 dwg, 9 tbl, 2 ex

Description

The invention relates to the field of biotechnology and can be used in agriculture and crop production for biological control, in the processing and disinfection of organic waste from pathogenic microorganisms.

Fungi of the genus Trichoderma are used in the biocontrol of root rot caused by phytopathogens of the genera Fusarium, Alternaria, Bipolaris. Among them there are also strains that can directly affect the growth and development of plants. This effect of strains of the genus Trichoderma on plants is due to their rapid colonization of the rhizosphere of plants, suppression of pathogens and saprotrophic microorganisms-competitors, improved seed germination, stimulation of the growth of the root system and increased plant immunity. They are used as a source of transgenes and herbicides, since the leading mechanism of their effect on plants is associated with the production of certain secondary metabolites [Zeilinger et al. 2016; Patle et al. 2018]. Representatives of the genus Trichoderma synthesize a number of plant growth stimulants - indoleacetic acid, ethylene, salicylic acid, compounds with antibiotic and phytotoxic effects [Zeilinger et al., 2016; Wu et al., 2018].

Strains of the species Trichoderma asperellum and T. koningii are known that synthesize biologically active secondary metabolites that inhibit pathogenic yeasts and bacteria (Reino et al. 2018).

Known strain Trichoderma erinaceum, isolated from tree bark in India, an agent of biocontrol and biofertilizer when growing rice, inhibiting phytopathogenic fungi Rhizoctonia solani, Sclerotium rolfsii, R. solani and Helmintosporium oryzae in the field [Swain et al., 2018).

Known strain GDFS1009 Trichoderma asperellum, which has antifungal activity against phytopathogens Fusarium oxysporum f.sp.cucumerinum and Fusarium graminearum - causative agents of wilting of cucumber and stem rot of corn [Wu et al., 2017].

Closest to the proposed invention in terms of the spectrum of biological activity, which should be taken as the closest analogue, is the strain Trichoderma asperellum MG-97 (VKPM F-765), which is used to accelerate the processing of organic waste by earthworms, which has antifungal to phytopathogens and plant growth-stimulating properties [RF Patent No. 2467989, priority dated 11/27/2012].

The disadvantages of the strain Trichoderma asperellum MG-97 (VKPM F-765) are low growth-stimulating activity and the degree of suppression of fungal infection of plants, a weak effect on the development of earthworms. For Trichoderma asperellum VKPM F-765, it is not known which compound (or compounds) is associated with its antibiotic and stimulating effect on organisms, which complicates its use.

The objective of the present invention is to isolate a strain of the microscopic fungus Trichoderma viride, which produces a plant growth stimulating substance - soyasaponin, with high activity against phytopathogenic and opportunistic fungi, which improves the development of the earthworms Eisenia fetida.

Saponins, derivatives of cyclopentanperhydrophenanthrene, triterpene compounds, and some authors also isolate steroid alkaloids synthesized by various organisms, which have a variety of effects on living organisms, in particular, can accelerate seed germination, plant growth and development, exhibit antimicrobial activity at certain concentrations [Paul et al. 2007; Wu et al., 2012, 2013; Butova, Salnikova, 2015].

The technical result of the invention is a new synthesizing soyasopanin I strain Trichoderma viride 346 (VKPM F-1525) with high growth-stimulating and antibiotic activity, including against microscopic mycelial and yeast fungi - opportunistic and phytopathogenic species, increases seed germination and plant growth intensity, reproduction and biomass accumulation by earthworms Eisenia fetida.

The essence of the invention lies in the fact that the new strain Trichoderma viride 346 (VKPM F-1525) is grown on Sabouraud's medium and medium supplemented with 3% unhopped wort with pH 7.2 under stationary conditions in 500 ml Eilenmeyer flasks for 14 days at 25 ° C. Antibiotic substances from the filtered culture liquid (CL) are extracted with ethyl acetate (in the ratio of ethyl acetate / CL 5: 1). The antibiotic activity of the culture fluid extracts was determined using the disk-diffusion method. On sterile paper disks ("Research Institute of Pasteur", Russia), 10 μl of extract with a concentration of 100 mg / ml is applied and dried under sterile conditions.

To assess the activity, collection test cultures of mycelial and yeast microscopic fungi are taken - opportunistic strains, including toxigenic micromycetes Aspergillus niger INA 00760, Candida albicans ATCC 2091, - A. oryzae 1K, A. niger, A. fumigatus 4K, A. terreus 4K, A. fisheri 3K, A. Flavus 7K, A.ustus 8K and phytopathogenic strain F. oxysporum VKM F-140. Standard disks with amphotericin B (Pasteur Research Institute, 40 μg / ml) are used as control.

The extracts inhibit the growth of all test cultures of filamentous fungi, but are especially effective against the phytopathogen F. oxysporum VKM F-140, common opportunistic strains A. fumigatus 4K, A. niger INA 00760, C. albicans ATCC 2091. Their activity against these test strains exceeds that of amphotericin B.

Flash chromatography is used to fractionate the primary extract. The active fractions are purified using reverse phase high performance liquid chromatography. The molecular weight of the active compound in the isolated fraction is determined by LC-MS / MS. The identity of the active compound to soyasaponin I is established by comparing the fragmentation spectrum of the isolated compound to the data for soyasaponin I described in the literature.

By introducing an aqueous suspension of spores and fragments of T. viride F-1525 mycelium (2 × 10 ⁶ CFU / kg) into the Eisenia fetida vermiculture on manure with straw, its productivity is increased. After the completion of vermicomposting, a larger (by 40%) number of worms are obtained, the biomass of adult worms - by 30%, and cocoons by 45%. When using this suspension when growing cucumbers, seed germination increases (by 5 days it is not less than 87%, and the length of plants increases by 28%, and the death of plants from root infection is significantly reduced or completely eliminated.

Strain Trichoderma viride 346 was isolated from soil (southern chernozem, Krasnodar Territory), from which a clone of group 2 was obtained by vegetative compatibility, which has a high growth rate and sporulation intensity.

Cultural and morphological characteristics of the strain. Colonies are rapidly growing, reaches 32-38 mm in diameter on the 6th day on Sabouraud's medium, the edge of the colony is sterile, mycelium is light white, even, conidial sporulation from lemon-yellow-green to dark green, but most often emerald in in the form of compact or loose bundles. There is no exudate. The reverse side of the colony is not colored or greenish. Good growth on wort - agar and Czapek medium. The hyphae are thin-walled, uncolored, 0.5-2.0 µm thick, often united in cords, abundant conidial sporulation. Conidiophores are mostly simple, often highly branched, ending in one or more phialids, conidiophores 20-30 microns long, conidia are most often smooth ellipsoid, 3-5 × 2-4 microns, globular conidia are rare, chlamydospores, sclerotia are absent.

Physiological and biochemical signs.

Strain grows on glucose-peptone agar, glucose-ammonium agar, wort-agar, Sabouraud's, Czapek's medium. The optimum growth temperature is 25-26 ° C. The strain assimilates well and moderately the following readily available carbohydrates - fructose, glucose, sucrose, glycogen, mannose, trehalose, xylose, as well as mannitol, acetic acid, gluconic, fumaric, ketoglutaric acids, adenosine and other organic sources of nitrogen (peptone) and other mineral sources (peptone.) And The strain produces active substances - soyasaponins, which have antimicrobial activity.

The strain is stored under a layer of vaseline on Czapek's medium (sucrose - 30 g, NaNO ₃ - 3 g, KH ₂ PO ₄ - 1 g, MgSO ₄ 7H ₂ O - 0.5 g, KCl - 0.5 g, FeSO ₄ 7H ₂ O - 0.01 g , agar-agar - 15 g) and in test tubes on Czapek's slant medium at 4 ° C with sowing after 0.5-1 year. For long-term storage, standard methods of lyophilization and cryopreservation are used.

Strain 346 was identified as Trichoderma viride based on sequencing of ITS regions (1-2) and 5.8S rDNA and cultural and morphological characters.

The nucleotide sequence ITS (1-2, including 5.8S) rDNA of strain 346 is registered in the Genbank under the number KC987155.1.

Similarity of the new Trichoderma viride PERS strain. VKPM F-1525 with collection strains of the species Trichoderma viride is 98.0%, which indicates along with its cultural and morphological properties that it belongs to Trichoderma viride. The strain is not genetically modified. The strain is non-pathogenic and non-toxic microorganism.

The Trichoderma viride 346 strain was deposited in the All-Russian Collection of Industrial Microorganisms under the number F-1525.

Strain Trichoderma viride PERS. VKPM F-1525 belongs to mitospore fungi - anamorphs of ascomycete affinity: genus Hypocrea, family Hypocreaceae, order Hypocreales, class Sordariomycetes, division Ascomycota (www.indexmngorum.org).

The invention solves the problem of expanding the range of biological products with antifungal and growth-stimulating activity and biological agents to accelerate the growth and reproduction of earthworms when composting organic waste.

The examples below illustrate the invention.

Example 1. Determination of the fungicidal activity of the Trichoderma viride PERS strain. VKPM F-1525 in relation to strains of opportunistic and phytopathogenic filamentous and yeast fungi

A 5-day culture of the fungus obtained on wort agar is used as an inoculum. The strain is grown on Sabouraud's medium and medium supplemented with 3% unhopped wort with pH 7.2 under stationary conditions in 500 ml Eilenmeyer flasks for 14 days at 25 ° C. The biomass of mycelium and spores is separated by centrifugation in a centrifuge. Antibiotic substances from the filtered culture liquid (CL) are extracted with ethyl acetate (in the ratio of ethyl acetate / CL 5: 1). Active compounds are secreted by the strain into the culture fluid.

(Швейцария) досуха при 42°С, сухой остаток - препарат хранят при 4°С. Для определения антибиотической активности используют диско-диффузионный метод [Егоров, 2006]. На стерильные бумажные диски («НИИ Пастера», Россия) наносят 10 мкл экстракта с концентрацией 100 мг/мл и высушивают в стерильных условиях.The resulting extracts are evaporated in vacuum on a rotary evaporator

(Switzerland) dryness at 42 ° С, dry residue - the preparation is stored at 4 ° С. The disc diffusion method is used to determine antibiotic activity [Egorov, 2006]. On sterile paper disks ("Research Institute of Pasteur", Russia), 10 μl of extract with a concentration of 100 mg / ml is applied and dried under sterile conditions.

To assess the activity, collectable test cultures of mycelial and yeast microscopic fungi of pathogenic strains Aspergillus niger INA 00760, Candida albicans ATCC 2091, opportunistic and toxigenic micromycetes - A. oryzae 1K, A. Niger INA 00760, A. fumigatus 4K, A. , A. fisheri 3K, A. Flavus 7K, A.ustus 8K and the phytopathogenic strain F. oxysporum VKM F-140. Standard disks with amphotericin B (Pasteur Research Institute, 40 μg / ml) are used as control. The size of the diameter of the zone of inhibition of the growth of test cultures is estimated at 5-7 days. Petri dishes with yeast are incubated at 37 ° C on Sabouraud's medium, with pathogenic and opportunistic microscopic fungi at 28 ° C on Czapek's medium.

Extracts of the culture fluid of T. viride strain F-1525 have a wide spectrum of fungicidal activity against opportunistic fungi. The extracts inhibit the growth of all test cultures of filamentous fungi, but are especially effective against the causative agent of root rot F. oxysporum VKM F-140, opportunistic strains A. fumigatus 4K, A. niger INA 00760, C. albicans ATCC 2091. Antibiotic the activity of extracts from the culture fluid of the strain in relation to other test objects in the overwhelming majority of cases exceeds the activity of amphotericin B (table 1).

Example 2. Isolation, separation of fractions and identification of individual antibiotic compounds (soyasaponins) of T. viride F-1525 strain

The T. virideF-1525 strain secretes antimicrobial substances - soesaponin into the culture fluid. To isolate antibiotic substances, the culture fluid (CL) of the producer is extracted with ethyl acetate in a ratio of ethyl acetate / CL of 5: 1. The obtained extracts are evaporated in vacuum on a Rotavapor-RBuchi (Switzerland) rotary evaporator to dryness at 42 ° C. The dry residue is dissolved in aqueous 50% ethanol and alcohol concentrates are obtained.

Fractionation of the antibiotic complex of the extract was performed using flash chromatography on silica gel (40-63 μm, column size 40 × 60 mm). The extract was eluted with a stepwise gradient of methylene chloride / methanol (v / v 50: 1, 20: 1, 10: 1, 5: 1, 3: 1, 100 ml each).

Sequential separation of the antibiotic complex of the extract from CL on silica gel by flash chromatography allows one to obtain 7 enriched fractions, in which the spectrum of antifungal activity against opportunistic fungi is assessed.

Two fractions F _II and F _III show the maximum activity against fungi, bacteria and yeast (Table 2), and have common components - soyasaponins (Figure 1).

For further purification after flash chromatography, fractions FII and FIII are subjected to fractionation by reverse phase high performance liquid chromatography (RP-HPLC) using a SunFire ™ C18 5μm 4.6 × 250 mm column (Waters, Ireland) in a linear gradient of increasing concentration the mobile phase created by eluent A (0.1% trifluoroacetic acid (TFA) in MQ water) and eluent B (80% acetonitrile with 0.1% aqueous TFA solution) at a flow rate of 1 ml / min. For RP-HPLC, ultra-gradient acetonitrile from Panreac (Spain) and TFA from Sigma-Aldrich (USA) are used. The detection of the substances to be separated is carried out at 214 nm in a gradient of the concentration of eluent B: 40-95% - for 20 minutes, followed by isocratic elution for 20 minutes.

The molecular weights of the active compounds in the isolated fraction are determined by liquid chromatography and tandem mass spectrometry LC-MS / MS. Mass spectrometric analysis determines the mass of the active compound - 942.5 Da, which coincides with the mass of soyasaponin I. The identity of the active compound to soyasaponin I is established by comparing their MS / MS fragmentation spectra for molecular ions obtained in the positive ion registration mode. Fragmentation spectra of soyasaponin I were obtained from the DEREPLICATOR + database (Mohimani et al., 2016). The coincidence of the fragmentation patterns of the isolated compound and soyasaponin I is illustrated in Figure 2. The content of soyasaponin in the culture fluid is 0.7 to 0.85 g / l of the medium.

Example 3. The effect of inoculation of the strain Trichoderma viride F-1525 into vermiculture on the accumulation of biomass and the number of earthworms Eisenia fetida

Sexually mature individuals of the same age of Eisenia fetida are used. The worms are kept in 5.5 liter plastic containers (height × length × width: 11 × 30 × 16 cm) with the lid open and the lights on to prevent them from crawling out. The substrate for them is prepared from cow dung and low-lying peat (pH = 7) with straw. Wheat straw is crushed on the KR-01 "Farmer-5" installation to a size of no more than 1 cm. 430 g of manure with peat, 100 g of straw and 1000 ml of distilled water are added to each container.

One variant of the experiment represents 3 containers with a mixture of substrates, into which 50 individuals of red Californian worms - Eisenia fetida are introduced. The second option is three containers with the same number of worms (50 individuals in a container) and strain T. viride F-1525 in an amount (1 × 10 ⁶ CFU / container or 2 × 10 ⁶ CFU / kg) in the form of an aqueous suspension of spores and mycelium ... Incubation (vermicomposting) is carried out for 60 days at room temperature (18-23 ° C). The moisture content of the organic substrate mixture is maintained at 80% of its total moisture capacity.

It is obtained that on the variant with the introduction of T.viride F-1525 into the vermiculture in comparison with the control (variant only with Eisenia fetida), the number of worms after the completion of vermicomposting is almost 40% higher, and the biomass of adult worms is higher by 30% (Table 3, four).

Example 4. Effect of inoculation of the Trichoderma viride F-1525 strain into vermiculture on the production of cocoons by the earthworms Eisenia fetida

The experiment is carried out according to example 6 with a number of differences. The composting mixture is made up of the following number of substrates: straw - 100 g, manure - 323 g, water - 857 g. Vermicomposting is carried out in a dark room. Trichoderma viride F-1525 is inoculated into moistened straw ((100 grams of straw + 700 ml of water + 1.5 × 10 ⁶ CFU T Viride F-1525) 5 days before preparing the mixture for vermicomposting and incubating it at room temperature. variant, straw moistened and incubated for 5 days is also introduced.After 60 days, cocoons are counted and it is obtained that their number in the variant with Trichoderma viride F-1525 is much higher (on average 183 in one container) compared to the control (without adding Trichoderma ) - 126 in one container (table 5).

Example 5. Activity of Trichoderma viride F-1525 in suppressing plant root rot disease

To assess the ability of Tviride F-1525 (titer 5 * 10 ⁵ CFU / kg of substrate with soil) to suppress phytopathogenic fungi, it is introduced as part of the formed vermicompost, obtained according to example 3, into sod-podzolic soil. The soil is sieved through a 1 mm sieve and mixed with vermicompost in a ratio of 1: 5 by volume. 400 ml of soil and 100 ml of vermicompost are added to rectangular containers, the mass with water is about 570 g. Cucumbers (15 seeds per container) and the Bouquet salad (30 seeds per container) are grown on an artificial infectious background F. oxysporum BKM F-140. The phytopathogenic strain of F. oxysporum BKMF-140 is introduced in an amount of 5 × 10 ⁶ CFU / g. Plants are incubated under artificial light, substrate moisture content of 35%, which is controlled by weight, and a temperature range of 18-24 ° C. Periodically take into account the death (lunge) of plants within 15 days (watercress) and 60 days for cucumbers.

It is obtained that the strain T. viride F-1525 significantly reduces the disease of Fusarium lettuce variety "Bouquet" (from 60% to 17%), and completely eliminates the death of cucumbers from Fusarium rot (table 6).

A similar experiment is carried out with another species of phytopathogenic fungus of the genus Fusarium, and a decrease in plant death by up to 15% is also obtained.

Example 6. Growth-stimulating effect of T.virideF-1525 on plants

The experiment is carried out with seeds of cucumber varieties "Farmer F1" in three repetitions (30 seeds in repetition). The seeds are moistened with water and placed on sod-podzolic soil in a container (control option), which is pre-moistened to an optimum moisture content of 60% of the total moisture capacity. In the experimental version, the seeds are moistened with an aqueous suspension of spores and mycelium with residues of culture liquid T. viride F-1525 (the titer of spores and mycelium fragments is 2 × 10 ⁶ CFU / kg soil). Incubation is carried out at constant humidity and light, and at room temperature. Take into account the germination of seeds and the length of the seedlings for 5 days. It is obtained that the germination of seeds in the control is 60%, and in the experiment - 93%. The average length of seedlings in the experimental variant is 22% higher than in the control variant.

Example 7. The effect of inoculation of the proposed strain T.viride F-1525 and the prototype strain Trichoderma asperellum VKPM F-765 on the accumulation of biomass and production of cocoons by earthworms Eisenia fetida

The experiment is carried out according to example 3. The control is the option without introducing strains into the Eisenia fetida vermiculture in a substrate with 80% moisture based on manure with the addition of peat and straw. Evaluate the effect of inoculation of the prototype strain Trichoderma asperellum VKPM F-765 and the proposed strain T.viride F-1525 in the vermiculture Eisenia fetida at the same dose (2 × 10 ⁶ CFU / kg substrate).

It is obtained that on the variant with the introduction of the proposed strain T.viride F-1525 into the vermiculture, the biomass of worms after the completion of vermicomposting is more than in the control (variant without inoculation of fungi) by 30%, and when using the prototype strain - only by 12%, then there is an increase in the accumulation of biomass of worms significantly higher in the case of T.viride F-1525.

The biomass of earthworms increased during vermicomposting in control by 28%, with Trichoderma asperellum by 43%, and by the proposed method with T. viride F-1525 by 58%.

When the prototype strain Trichoderma asperellum BKPMF-765 is inoculated, the production of cocoons in the Eisenia fetida vermiculture increases on average by 20% compared to the control. The number of cocoons in the variant with the proposed strain TvirideF-1525 is significantly higher (by 45%) than in the control, and exceeds that of the prototype strain.

Example 8. Growth-stimulating activity of the proposed strain T. virideF-1525 and prototype strain Trichoderma asperellum VKPM F-765 on cucumbers

The experiment is carried out according to example 3. Take into account the germination of seeds and the length of the seedlings for 5 days. Get that seed germination in the control 55%, with the prototype strain - 55%, the proposed strain - 87%. The average length of cucumber seedlings during inoculation of the prototype strain increases by 13% compared to the control, and the proposed strain by 28%, that is, significantly more (table 8).

Example 9. The ability of the proposed strain T.viride F-1525 and the prototype strain Trichoderma asperellum VKPM F-765 to suppress the development of fusarium rot of cucumber roots

The experiment is carried out according to example 5. Evaluate the death of plants of cucumber varieties "Fermenter F1" when grown on a fusarium infectious background.

It is obtained that in the case of inoculation of the prototype strain Trichoderma asperellum BKPMF-765, the death of cucumbers from root rot is 10 and 22% on 10 and 30 days, respectively, and in the case of using the proposed strain, cucumbers did not die during this period.

Bibliography

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RF patent No. 2467989, priority dated November 27, 2012.

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Claims (1)

The strain of the microscopic fungus Trichoderma viride VKPM F-1525 is a producer of soyasaponin I, which has growth-stimulating activity against plants and earthworms Eisenia fetida and antimycotic effect against opportunistic and phytopathogenic fungi.

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