Trichoderma pseudokoningii strain that responds to strigolactone mimics

Abstract

The present invention relates to a strain of Trichoderma pseudokoningii Tpk20, deposited under DSM number 34838 at Leibniz-lnstitut DSMZ - German Collection of Microorganisms and Cell Cultures, which has a biostimulant action on plants, due to the specific production of compounds that increase plant nutrients availability and / or activate plant metabolic defense pathways, and which responds to strigolactone mimics by amplifying the production of compounds involved in the plant biostimulant effects.

Description

[0001] TRICHODERMA PSEUDOKONINGII STRAIN THAT RESPONDS TO STRIGOLACTONE MIMICS

[0002] The present invention relates to a strain of Trichoderma pseudokoningii which has a biostimulant action on plants, due to the specific production of compounds that increase plant nutrients availability and / or activate plant metabolic defense pathways, and which responds to strigolactone mimics by amplifying the production of compounds involved in the plant biostimulant effects.

[0003] Different strains of Trichoderma biostimulant effects are known to be effective on cultivated plants. Patent application WO2022029343 A1 refers to a T. harzianum biostimulant strain, deposited at the Spanish Collection of Type Crops (CECT) with deposit number CECT21179, and its use to increase the fertilizers use efficiency. Patent EP3764797 B1 describes a fungal strain of the genus Trichoderma, named HSA12 and deposited under DSM number 32722 at the Leibniz-lnstitute DSMZ - German Collection of Microorganisms and Cell Cultures. This fungal strain increases the tolerance of tomato, corn, and soybean plants to abiotic stresses — water stress, low-temperature stress, and oxidative stress. The HSA12 strain is also effective for the remediation of polluted soil and water, as well as for ecological restoration, including the recolonization of plant populations that are endangered or valuable for a particular environment.

[0004] Patent FR2903419 B1 protects the strain deposited under MUCL number 45632 at the Belgian Coordinated Collections of Microorganisms (BCCM), which belongs to the species T. atroviride, and which stimulates plant germination and growth.

[0005] The US Patent 8716001 B2 describes the use of strains of Trichoderma, T. atroviride WW10TC4 (deposit number ATCC PTA 9707), T. harzianum RR17Bc (deposit number ATCC PTA 9708), T. harzianum F11 Bab (deposit number ATCC PTA 9709), applied separately or in combination thereof, to inducing plant resistance to biotic and abiotic stresses; reducing the negative impact of excess nitrate (emissions of nitrous oxide from the soil, leaching of nitrate ions into the aquifer and surface water) due to increased efficiency of nitrogen use by plants; increasing the amount of carbon sequestered from the atmosphere due to increased photosynthesis processes in plants whose rhizosphere is colonized by those strains.

[0006] Patent US8598083 (B2) refers to the strain of Trichoderma harzianum TSTh20-1 , deposited under number PTA-10317 at the American Collection of Cultures of Microorganisms (ATCC), which can stimulate plant growth, under conditions of abiotic stress caused by the presence of polycyclic aromatic hydrocarbons, naphthenic acids and high pH, specific to tailings left after heavy crude oil extraction from tar sands.

[0007] Patent EP1990404 B1 discloses the T. atroviride AGR2 strain, deposited at the National Collection of Microorganisms Cultures (CNCM), Pasteur Institute, under number 1-2738, and claims its uses as a germination, root, and plant growth promotor.

[0008] Strains of Trichoderma in which the plant biostimulant activity, including on plant growth and development, is amplified by exo-signals secreted by plant roots in the rhizosphere are effective in conservative agriculture (CA) I high plant residue systems. These CA systems conserve environmental resources and reduce inputs into agricultural technologies by eliminating / reducing tillage. In CA systems, loosening and structuring the soil necessary for germinating and developing crops is achieved by intensifying soil biota activity, favored by maintaining plant residues at the soil surface. Permanent soil coverage with plant residues is an essential conservation agriculture (CA) system principle. Plant residues covering the soil limit the evaporation of water and facilitate its infiltration, reduce erosion, improve soil structure, increase organic matter and carbon content, and moderate soil temperature in warm areas (Fabrizzi et al. 2005, Soil and Tillage Research, 81:57-69). Despite the many advantages, several adverse effects are also associated with high residue systems. Plant residues promote the development of soil phytopathogens (Bockus and Shroyer 1998, Annual Review of Phytopathology, 36: 485-500), including devastating ones such as those that cause Fusarium head blight (Leplat et al. 2013, Agronomy for Sustainable Development, 33: 97-111).

[0009] The strain T. pseudokoningii Td85 was deposited at the Leibniz-lnstitute DSMZ - German Collection of Microorganisms and Cell Cultures (DSMZ) under deposit number DSM 23661 and is intended for use in conservative farming systems. The strain, protected by patent RO127471 B1, exhibits concomitant antagonism to soil phytopathogens, high plant residues mineralization capacity, and resistance to biofumigant compounds released from cruciferous biomass, being planned to be used mainly for CA systems using the biofumigation properties of cruciferous crops. However, an amplification of the production of compounds involved in biostimulants and / or antagonistic activities under the action of exo-signals secreted by stressed cultivated plants / their root system has not been claimed for this strain.

[0010] An example of exo-signals with a role in forming mutualist associations are strigolactones. Strigolactones are carotenoid compounds synthesized by plart roots, with the role of exo- and endo-signals, respectively, semiochemicals that mediate interactions in the rhizosphere, and (phyto)hormones, which control physiological processes in plants (Cavar et al. Phytochemistry Reviews, 2015, 14, 691-711). In recent years, the existence of canonical strigolactones, which have three condensed cycles (ABCs) linked by an etheric group by a furanonic a,p~unsaturated cycle (D), usually substituted by one or two methyl radicals, and non-canonical ones, which have an open BC cycle (Kee et al. 2023, Plant and Cell Physiology, 64(9), 955-966).

[0011] Due to the complexity of natural strigolactones and the difficulty of obtaining them in quantities sufficient for practical application, analog compounds have been synthesized, which include a furan-2-one cycle and a typical lactone structure with three strigolactone-specific aromatic cycles, or strigolactone mimics, which have some of the biological activities specific to strigolactone, include a furan-2-one cycle, but without the typical structure with the three aromatic cycles (Zwanenburg et al. Plant, 2016, 243, 1311-1326). Strigolactone analogs follow the molecular logic of canonical strigolactones, and strigolactone mimics have a chemical structure that starts from non- canonical strigolactones.

[0012] Till now only one strain of Trichoderma, T. asperellum Td36b, deposited under the number P(F) 001434 at National Collection of Agricultural and Industrial Microorganisms (NCAIM) Budapest, has been described that responds to several compounds, which plants secrete as rhizosphere exo-signals or their synthetic analogs / mimics, including the strigolactone analog GR-24, by amplifying the biosynthesis of metabolites that increase plant nutrients bioavailability and / or promote plant growth and development, and / or controls the development of fungal phytopathogens (Patent RO131177 B1).

[0013] However, till now, there were no described biostimulant strains of Trichoderma acting complementary to strigolactone analogs and mimics in reducing stress (especially water stress) in tomato plants grown in the field conditions and promoting the accumulation of beneficial compounds in tomato fruits, such as lycopene and gamma-amino-butyric acid.

[0014] In tomato plants grown in the field, deficit irrigation is one of the strategies to reduce production costs, especially in field conditions (Valcarcel et al., 2020, Scientia Horticulturae, 261, 108972). Still, it has the disadvantage of having an elevated risk for an economically significant reduction in the production level. (Khapte et al. 2019, Scientia Horticulturae, 248, 256-264). Application of GR24, an strigolactone aaafag, facilitated the recovery of tomato plants after water stress (Visentin et al. 2020, Plant, Cell & Environment, 43(7), 1613-1624). Biostimulant strains of Trichoderma also increase the water stress tolerance of tomatoes (Rawal et al. 2022, Frontiers in Plant Science, 13, 869090.) Using strains of Trichoderma and strigolactone mimics and / or analogs is a solution to ensure economically profitable tomato production levels under poor irrigation conditions.

[0015] Until now, no patented strains of Trichoderma were claimed to stimulate the accumulation of bioactive compounds in tomato fruits. However, there are studies demonstrating that the application of Trichoderma biostimulant strains determines an increase in the accumulation of lycopene and gamma-amino-butyric acid in tomatoes (Carillo et al. 2020, Plants, 9(6), 771). Until now, no Trichoderma strains were demonstrated to interact complementarily with strigolactone analogs and / or mimics in stimulating bioactive compounds, lycopene, and gamma-amino-butyric acid accumulation in tomato fruits produced by treated plants.

[0016] Strain of T. pseudokoningii Tpk20, according to the invention, deposited under DSM number 34838 at Leibniz-lnstitute DSMZ - German Collection of Microorganisms and Cell Cultures, Braunschweig, has an antagonistic effect towards phytopathogens that develop on plant debris, promote plant nutrition by producing compounds that solubilize phosphates and siderophores, biosynthesizes cellulolytic systems that also include non-catalytic proteins of swolenin type, that loosen the fibrillar structure of cellulose and promote root colonization, increases the efficiency of water use in tomato plants grown in the field, activates secondary metabolism in treated tomato plants, promoting the accumulation of lycopene and gamma-amino-butyric acid, responds to the strigolactones mimics SL-20, 2-(4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)-6-(4- methylpiperidin-1-yl)-benzo[de]isoquinoline-1, 3-dione, and SL-21 , 2-(4-methyl-5-oxo- 2, 5-dihydro-furan-2-yloxy)-6-(4-benzyl-piperidin-1-yl)-benzo[de]isoquinoline-1 ,3-dione, by amplifying the production of phosphorus-solubilizing compounds, siderophores, and cellulase systems that include non-catalytic swolenin proteins, it acts complementarily with strigolactone mimics on field-grown tomato plants in terms of increasing water use efficiency and accumulating lycopene and gamma-amino-butyric acid in fruits.

[0017] In the strain of T. pseudokoningii Tpk20 described in this invention, the production of phosphate-solubilizing compounds is amplified on average by 72.3% and, respectively, 82.8%, by the presence in the culture medium of the strigolactone mimics, SL-20, 2-(4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)-6-(4-methylpiperidin-1-yl)-bfinzo[del izochinolin-1 , 3-dione and, respectively, SL-21 , 2-(4-methyl-5-oxo-2,5-dihydro-furan-2- yloxy)-6-(4-benzyl-piperidin-1-yl)-benzo[de]isoquinoline-1 ,3-dione, in concentration of 10-7M.

[0018] In the strain of T. pseudokoningii Tpk20 described in this invention, the production of siderophores is amplified on average by 45.3% and, respectively, 63.8%, by the presence in the culture medium of the strigolactone mimics, SL-20, 2-(4-methyl- 5-oxo-2,5-dihydrofuran-2-yloxy)-6-(4-methylpiperidin-1-yl)-benzo[de]izochi-nolin-1 ,3-dione and, respectively, SL-21 , 2-(4-methyl-5-oxo-2,5-dihydro-furan-2-yloxy)-6-(4-benzyl- piperidin-1-yl)-benzo[de]isoquinoline-1 , 3-dione, in concentration of 10-7M.

[0019] In the case of the T. pseudokoningii Tpk20 strain described in this invention, the production of cellulolytic systems that also include non-catalytic proteins of swolenin type that loosen the fibrillar structure of cellulose and promote colonization of the rhizoplane is amplified on average by 83.5% and 72.6%, respectively, in the presence in the culture medium of strigolactone mimics, SL-20, 2-(4-methyl-5-oxo-2,5-dihydrofuran- 2-yloxy)-6-(4-methyllpiperidin-1 -yl)-benzo[de]izochi-nolin-1 ,3-dione and respectively, SL-21 , 2-(4-methyl-5-oxo-2,5-dihydro-furan-2-yloxy)-6-(4-benzyl-piperidin-1 -yl)-benzo [de]isoquinoline-1 ,3-dione, at a concentration of 107M.

[0020] In the case of T. pseudokoningii strain Tpk20, when applied foliar at a dose of 1012chlamydospores / ha, together with strigolactone mimics, SL-20, 2-(4-methyl-5-oxo- 2, 5-dihydrofuran-2-yloxy)-6-(4-methylpiperidin-1-yl)-benzo[de]isoochi-nolin-1 ,3-dione and, respectively, SL-21 , 2-(4-methyl-5-oxo-2,5-dihydro-furan-2-yloxy)-6-(4-benzyl- piperidin-1-yl)-benzo[de]isoquinoline-1, 3-dione, applied as soil treatment, promotes yield in deficit irrigation conditions and determines a significantly increased accumulation of lycopene and gamma-amino-butyric acid in tomato fruits compared to the untreated control, or experimental variants treated with only one of the components, Tpk20 strain or strigolactone mimics.

[0021] Further examples of invention embodiments are presented below, illustrating it without limiting it.

[0022] Example 1. Strain T. pseudokoningii Tpk2Q, deposited under DSM number 34838 at Leibniz-lnstitute DSMZ - German Collection of Micro-organisms and Cell Cultures, Braunschweig, was isolated from the soil liter, sampled from Breaza - Valea Tarsei area, Prahova county, Romania. A water-agar culture medium was used for isolation, and the potato-glucose-agar (PDA) medium was used to purify isolates. The radius of colonies developed by the T. pseudokoningii Tpk20 strain on CGA medium after 72 hours, at 30°C is (12-) 48 (-64) mm; at 35°C it is (0-)16(-24) mm and at 37°C: 0 mm. This inhibition at 37°C involves a low pathogenicity for humans and warmblood animals whose internal temperature is 37°C. Colonies formed on the CGA medium at 25°C after 40 hours in darkness do not show any yellow pigment diffusing in the agar medium. Colonies grown for 72 hours on PDA medium, at 30°C, in the dark, form up to 7 concentric rings, with a dense production of conidia and without aerial mycelium. The conidia are dark green towards the center, and they are only forming towards the edges. The conidia formed are dark green, globular to sub-globular or ovoid, with sizes of 4.0-5.0 (-6.0) * 2.5-3.0 pm. Conidiophores have a symmetrical appearance ending in two or more phialides, with primary branches occurring near the apex, frequently paired and projected nearly 90 degrees from the central axis. Chlamydospores are abundantly formed after incubation for one week at 20 °C in the dark or after irradiation with red light (LED or laser). Chlamydospores are terminal and sometimes interspersed on immersed hyphae, sub-globular to ovoid, smooth, pale green. Phialides are typically produced at the tips of primary, secondary, and tertiary branches, rarely directly along the length of the branches, usually 2 to 4 phialides per whorl.

[0023] Physiological characteristics and use of various substrates are described below. Monosaccharides determine better growth than disaccharides, followed by polysaccharides. Glycerin is favorable for the growth of the Tpk20 strain. Among monosaccharides, ribose and fructose are the best carbon sources, with optimal strain development observed on media containing these carbon sources. Ribose and fructose are followed by glucose, mannitol, D-mannose, D-galactose, and L-arabinose, which allow moderate development. Among disaccharides, the best results were for lactose, followed by maltose and sucrose (which determine poorer development). Among polysaccharides, cellulose gave the best results, with optimal development, followed by starch, with moderate growth in media containing starch as a sole carbon source. The sporulation was very good in most variants (+++), good in the case of sucrose (++), and worse in the case of glycerin (+).

[0024] Peptone, amino acids L-asparagine, L-valine, L-serine, L-cysteine, L-isoleucine, NH4H2PO4, and NaNO2, are the best nitrogen sources, determining optimal development. Next in descending order are nitrates (NH4NO3, NaNOs), vitamin B12, and amino acids L-alanine, L-lysine, L-arginine, and L-tryptophan, which caused moderate development. Worse results had urea, NH4CI, KNO3, and KNO2. Sporulation was very good (+++) in most variants, except L-lysine, L-tryptophan, L-alanine, L- arginine, and nitrates, whereas sporulation was only good (++).

[0025] The growing temperatures are: optimum temperature: 22-25°C; minimum temperature: 2 °C; maximum temperature: 35°C. Temperatures between 10 and 18°C cause poor growth of Trichoderma pseudokoningii Tpk20, no sporulation at 48 hours and weak sporulation at 144 hours (+). Reaction of the growing substrate: optimal pH: 4.0-5.5; poor fungus development at pH values from 9.0 to 13.0.

[0026] The identification was achieved through a polyphase approach. A first identification was made using the Microbial ID Biolog system (Biolog, Hayward, CA, USA). This initial morphological identification was confirmed by molecular analysis of the ITS1 (internal transcribed spacers 1) cluster for the rRNA gene (BarCode universal fungal marker, http: / / www.isth.info). ITS1 SR6Rf and LR1r specific primers were used (according to BarCode protocol http: / / www.isth. info / methods). The sequences were compared with the TrichoBlast program (http: / / www.isth.info / tools / blast / index.php).

[0027] The Tpk20 strain is strongly antagonistic to the following phytopathogenic fungi: Rhizoctonia solanii, Fusarium graminearum, F. culmorum, Pythium ultimum, Botrytis cinerea, Altemaria alternata, Sclerotinia sclerotiorum. The degree of antagonism of the T. pseudokoningii T pk20 strain against phytopathogenic fungi was determined in vitro by double culture method (Coskuntuna and Ozer, 2008, Crop Protection, 27, 330-336). T. pseudokoningii Tpk20 strain and the tested phytopathogens (Rhizoctonia solanii ATCC 66873, Fusarium graminearum DSM4527, F. culmorum ATCC 36017, Pythium ultimum, DSM 62987, Botrytis cinerea DSM 5144, Altemaria alternata DSM 62010, Sclerotinia sclerotiorum DSM 1946) were grown separately on potato-glucose-agar medium (CGA) for 7 days at 25°C and in the dark. From mycelium formed after 7 days, 0.5 mm discs were taken with a drive pin punch. The mycelium discs were deposited 1 cm from the walls of a 9 cm diameter petri dish, on opposite sides, each disc being separated by approx. 6 cm of agarized medium. The interaction between colonies was determined after 5 days of incubation at 25°C and in the dark, using the scale proposed by Bell et al. (Bell et al., 1982, Phytopathology, 72, 379-382), with 5 notes: 1- Trichoderma strain grows over phytopathogen strain, covering the entire surface; 2 - Trichoderma strain grows on more than two-thirds of the growing medium; 3 - Trichoderma strain and the pathogen each grows on about half the surface of the medium; 4 - phytopathogen grows on more than two-thirds of the growing medium; 5 - the phytopathogen grows over Trichoderma, covering the entire surface The experiments were conducted in triplicate and repeated three times.

[0028] The analysis of the experimental results obtained by in-vitro antagonism tests (Table 1), demonstrated that T. psedukoningii Tpk20 strain shows marked antagonism to Rhizoctonia solanii ATCC 66873, Fusarium graminearum DSM4527, F. culmorum ATCC 36017, Pythium ultimum, DSM 62987, Botrytis cinerea DSM 5144, Altemaria alternata DSM 62010, Sclerotinia sclerotiorum DSM 1946.

[0029] Tab. 1. In-vitro antagonism of the T. psedukoningii Tpk20 versus phytopathogens.

[0030] Other experiments were intended to bioassay the biostimulant effect of the Tpk20 strain on tomato seedlings. Tomato seeds were disinfected in two stages. The first disinfection was conducted in 70% ethanol for 30 seconds, stirring at 60 rpm. After removing the ethanol, the seeds were rinsed three times with sterile distilled water. The second disinfection was done with 4% sodium hypochlorite solution for 15 minutes. Subsequently, rinses with distilled water were carried-out every 25 minutes for two hours. The seeds were inoculated by immersion in 3 ml suspension of fungal propagules at a concentration of 106cfu / ml in phosphate buffer with 2 % carboxymethylcellulose and then deposited in sterile Cyg growth bags (Mega International, Newport, MN, USA). The bags were moistened daily with a 0.25% Hoagland nutrient solution throughout the experiment. The root growth of tomato seedlings was analyzed at 3 weeks versus a control that was not inoculated. The results revealed that the Tpk20 strain significantly increased significantly in tomato test plant roots compared to the uninoculated control of over 162.3±14.2%. To detect the production of phosphorus-solubilizing compounds, a medium containing per 1 liter was used: glucose 10 g; (NH4)2SO4 0,5 g; NaCI 0,2 g; MgSO4.7H2O, 0.1 g; KCI 0.2 g; yeast extract 0.5 g; MnSO4.H2O 0,002 g and FeSO4.7H2O 0,002 g. (Reagents from Merck). 5 g of hydroxyapatite, Ca10(PO4)e(OH)2 (Sigma), was also added to this medium. The medium was sterilized by autoclaving (121 °C, for 20 min), cooled and distributed in Petri dishes 0 9 cm. The plates were inoculated with the Tpk20 strain and incubated for 5 days at 25°C. After 5 days, lysis zones around T . pseudokoningii Tpk20 colonies were found, demonstrating phosphorus-solubilizing compounds’ production.

[0031] The ability of the Tpk20 strain to produce siderophores was also detected. To detect the production of siderophores, an agar medium with complexation indicators (CAS blue agar) prepared from three solutions was used as detector medium: Solution 1 , 0,06 g Chrome Azurol S (CAS) in 50 ml of double distilled water in quartz plant; Solution 2, 0.027 g of FeCl3xeH2O in 100 ml of 10 mM HCI; Solution 3, 0.073 g hexadecyltrimethylammonium bromide (HDTMA) in 40 ml of double-distilled water. The solution (1) was mixed with 10 ml solution (2) and then was added solution (3). The resulting solution was sterilized by autoclaving at 121 °C for 15 minutes to obtain an axenic medium with blue iron complexes. A quantity of 32.24 g 2,2’-(Piperazine-1 ,4- diyl)diethanesulfonic acid (PIPES) was dissolved in 850 ml of double-distilled water, whose pH was brought to a value greater than 6. The resulting solution was mixed, after pH correction to 6.8 by adding 0.1 M NaOH, was agarized with 1.5% agar. The final solution was autoclaved and cooled to 50°C. The 100 ml of iron complex solution, brought to the same temperature, was added, aseptically and slowly, by draining on the wall of the glass vessel and stirring over the medium with PIPES, agarized, and sterilized. The reagents used to prepare the CAS blue agar detector medium came from Sigma-Aldrich (St. Louis, MO, USA).

[0032] The medium malt-agar extract (MEA) was distributed in Petri dishes 0 9 cm. Half of the MEA medium has been cut off and removed aseptically. The space thus freed up was replaced by an agarized detector medium prepared as above (CAS blue agar). The half MEA medium was inoculated with strain Tpk20. After incubation for 6 days at 25°C, the color of the medium changed from blue to orange, which is evidence of siderophore production.

[0033] The Tpk20 strain has been cultivated on a liquid medium to establish its ability to produce harzianic acid. This siderophore stimulates plant growth and development and has an anti-fungal effect (Vinale et al. 2009, Journal of Natural Products, 72, 2032- 2035). A decoction of potatoes was prepared by boiling 200 grams of potatoes, thoroughly washed, but not peeled, and cut into cubes with sides approx. 1 cm, with approx, one liter of distilled water for 30 minutes. The decoction was filtered through a double gauze cloth and added 20 grams of glucose (Sigma-Aldrich). The medium was brought to 1000 ml with distilled water, sterilized by autoclaving for 20 minutes at 121 °C and distributed in 5-litre conical vials. The potato-glucose liquid medium was inoculated with 50 ml containing 108cfu / ml propagules from the Tpk20 strain. The culture was maintained under stationary conditions for 21 days at 25°C. After 21 days, the culture medium was filtered through filter paper (Whatman No. 4, Brentford, UK).

[0034] The filtered culture medium was acidified to pH 4 with a 5 N HCI solution and extracted repeatedly with ethyl acetate (Merck, Darmstadt, Germany). The organic fractions were evaporated in vacuum at 35°C and dried on anhydrous sodium sulfate (Sigma Aldrich). The red residue was recovered in chloroform (Merck, Darmstadt) and extracted three times with a 2 N NaOH solution. An acid fraction containing harzianic acid was precipitated from the alkaline extract by treatment with HCI 2 N. In the acid fraction the content of harzianic acid was determined using the method described by Vinale et al. 2014, (Molecules, 19, 9760-9772). Production of 118 mg of harzianic acid per 1 liter of liquid potato-glucose medium incubated for 21 days was detected. This quantity is close to that determined for T. harzianum M10 strain in which the production of harzianic acid was first recorded (Vinale et al. 2009, Journal of Natural Products, 72, 2032-2035).

[0035] An experiment was conducted to determine oxygen consumption and the release of various compounds from plant material treated with the strain of T. pseudokoningii Tpk20 compared to strain T. harzianum Td50b, which has a high degradation capacity of plant material (EP patent application 2735607 A1). The plant material (dried and chopped hairy vetch stalks) was ground and passed to a 0.250 mm sieve. 10 g of powder were packed and sterilized by gamma irradiation (at IRASM, IFIN-HH, Bucharest, Romania). 0.1 g of powder of plant material was aseptically taken from the sterilized powder, which was brought into a sterile 50 ml Erlenmeyer. 19 ml sterile phosphate buffer was added aseptically to the finely ground powder, homogenized by shaking and inoculated with 1 ml microbial suspension, normalized to 108cfu (propagules) / ml. It was kept on the shaker for 24 hours at 28°C, after which it was aseptically passed into a Strathox breathing vessel (Strathkelvin Instruments Limited. Glasgow, UK). Determinations of carbon dioxide production were done for 12 hours. After performing breath determinations, supernatants were separated by filtration, in which Total Organic Carbon (TOC) was determined with a Formacs HT apparatus (Skalar Analytical B.V., Breda Netherlands), reducing carbohydrates (with DNS reagent) and total soluble phosphorus (with ammonium molybdate and stannous chloride reagent). The experiments were done compared to an uninoculated control.

[0036] The results are presented in Table 2. These results demonstrate a remarkably high mineralization activity of plant material for the Tpk20 strain.

[0037] Tab. 2. Degradation activity of plant material by assessed strains of microorganisms.

[0038] Example 2. The influence of strigolactones mimics SL-20, 2-(4-methyl-5-oxo-2,5- dihydrofuran-2-yloxy)-6-(4-methylpiperidin-1 -yl)-benzo[de]isoquinoline-1 ,3-dione and SL-21 , 2 -(4-methyl-5-oxo-2,5-dihydro-furan-2-yloxy)-6-(4-benzyl-piperidin-1-yl)- benzo[de]iso-quinoline-1, 3-dione, endo-signals that elicit the plant response under nutrient deficiency conditions or the action of biotic and abiotic stressors and / or exosignals that stimulate the formation of mutualist rhizosphere associations, on the characteristics of strain Tpk20 relating to the production of compounds that promote plant nutrients, i.e. compounds that solubilize inorganic phosphorus and siderophores, was determined.

[0039] A medium containing glucose 10 g; (NFU^SO 0.5 g; NaCI 0,2 g; MgSCUxThW, 0.1 g; KCI 0.2 g; yeast extract 0.5 g; MnSCkxfW 0.002 g and FeSO4x7H2O 0.002 g. (Reagents from Merck) was used to determine the influence of the presence of compounds with the role of exo-signals of rhizosphere (strigolactone mimics) on the solubilization capacity of phosphorus. The medium was supplemented with 5 g hydroxyapatite, Caio(P04)e(OH)2 (Merck), homogenized, and distributed 100 ml each into 500 ml Erlenmeyer jars. The Erlenmeyer jars, plugged with a cotton wool stopper, were sterilized by autoclaving (121°C for 20 min) and cooled. In the samples tested for the influence of compounds acting as rhizosphere signals, mimics of strigolactone, SL- 20, 2-(4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)-6-(4-methyllpiperidin-1-yl)- benzo[de]isoochi-nolin-1, 3-dione and, respectively, SL-21, 2-(4-methyl-5-oxo-2,5- dihydro-furan-2-yloxy)-6-(4-benzyl-piperidin-1-yl)-benzo[de]isoquinoline-1 ,3-dione, at a concentration of 10-7M, were introduced. The Erlenmeyer jars were inoculated with the Tpk20 strain and incubated for 5 days at 25°C. After the end of the incubation period, samples were taken from the culture media, clarified by ultrafiltration, and the level of soluble phosphorus was determined in the supernatant using the phosphomolybdic reagent method (Murphy and Riley, 1962, Anal. Chim. Acta 27:31-36.).

[0040] The production of siderophores is amplified in the environment by 45.3% and 63.8%, respectively, by the presence in the culture medium of mimics of strigolactone, SL-20, 2-(4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)-6-(4-methylpiperidin-1-yl)-benzo[de] isoochi-nolin-1 ,3-dione and, respectively, SL-21, 2-(4-methyl-5-oxo-2,5-dihydro-furan-2- yloxy)-6-(4-benzyl-piperidin-1-yl)-benzo[de]isoquinoline-1 ,3-dione, at concentrations of 107M.

[0041] The strain Tpk20 was cultured on liquid potato-glucose medium prepared as shown in Example 1, in the presence and absence of strigolactone mimics, SL-20, 2-(4- methyl-5-oxo-2,5-dihydrofuran-2-yloxy)-6-(4-methylpiperidin-1-yl)-benzo[de]isoochi- nolin-1 ,3-dione and, respectively, SL-21, 2-(4-methyl-5-oxo-2,5-dihydro-furan-2-yloxy)- 6-(4-benzyl-piperidine-1-yl)-benzo[de]isoquinoline-1 ,3-dione, at concentrations of 107M. Production of siderophores by strain Tpk20, in the presence and absence of SL-20, and, respectively, SL-21 , in potato-glucose medium incubated for 6 days at 25°C was determined with CAS reagent (Schwyn and Neilands, 1987, Analytical Biochemistry, 160, 47-56).

[0042] The production of phosphorus-solubilizing compounds by the T. pseudokoningii strain TpK20 is amplified on average by 72.3% and 82.8%, respectively, by the presence in the culture medium of the strigolactone mimics, SL-20, 2-(4-methyl-5-oxo- 2, 5-dihydrofuran-2-yloxy)-6-(4-methylpiperidin-1-yl)-benzo[de]isoochi-nolin-1 ,3-dione and respectively, SL-21, 2-(4-methyl-5-oxo-2,5-dihydro-furan-2-yloxy)-6-(4-benzyl- piperidin-1-yl)-benzo[de]isoquinoline-1, respectively, 3-dione, in concentration of 10-7M.

[0043] Example 3. The enzymatic activity of the strain was determined T. pseudokoningii Tpk20, obtained by growing on basal liquid medium supplemented with cellulose 2%. 1 liter of basal medium contains: 5 g ammonium tartrate, 1 g KH2PO4, 0,5 g MgSO4*7H2O, 0,1 g yeast extract, 0,001 g CaCl2.2H2O - 0,1 ml of a stock solution 10 mg / ml, to which was added 2% bacterial cellulose with an average molecular weight of 500 kDa. On this medium, sterilized by autoclaving for 20 minutes at 121 °C, T. pseudokoningii Tpk20 strain was cultivated for 21 days. The supernatant was recovered after centrifugation at 5,000 x g at 4°C for 20 min. The total protein in the extract was determined according to the Bradford method, using bovine serum albumin as standard. The enzyme activity was determined using Whatman filter paper No. 1 as substrate.

[0044] The filter paper was cut into 1 x 6 cm strips, from which 50 mg were taken and suspended in an 18 mm 1 10 ml test tube with 1 mL citrate buffer - sodium 0.05 M, pH 4.8. Over the filter paper - buffer mixture was added 0.5 ml of extract. It was vortexed for effective suspension of filter paper and incubated for 1 h at 45°C. After the incubation period, the reaction was stopped with 3 ml dinitrosalicylic reagent DNS (containing 283.2 ml deionized water, 2.12 g 3.5 dinitrosalicylic acid, 3.96 g NaOH, 61 .2 g sodium potassium tartrate, 1.52 ml 89% liquid phenol, and 1.66 g sodium bisulfite). The test tube was heated in a boiling water bath for 5 minutes to develop the color reaction, after which the optical density was read at 545 nm. Enzyme activity was expressed using a glucose standard curve in FPU units (Ghose, 1987, Pure Appl. Chem, 59, 257-268). One FPU unit is 0.37 I enzyme activity releasing equivalent to 2 mg glucose * enzyme units (pmoles reducing groups reacting with released DNS per min). The Tpk20 strain produced 0.37±0.07 FPU per mg protein under the test conditions, which means that it is a strain producing a high cellulolytic system (Kovacs et al., 2008, Enzyme and Microbial Technology, 43, 48-55).

[0045] The influence on the surface tension of water was also determined using an optical tensiometer OCA 50EC (Data Physics, Filderstadt, Germany). A meaningful change in the surface tension of water was found, demonstrating the existence in the cellulase system secreted by the TPk20 strain of non-catalytic proteins of the swolenin type, which are known to influence the water surface tension. (Frischmann et al. 2013, Journal of Biological Chemistry, 288, 4278-4287). These non-catalytic amphiphilic proteins of the swolenin type break down the fibrillar structure of cellulose by disruption of hydrogen bonds between cellulose fibrils (Zhou et al. 2011, World Journal of Microbiology and Biotechnology, 27, 1905-1910) and promote the colonization of plant tissues (Brotman et al. 2008, Plant Physiology, 147, 779-789).

[0046] The influence of introduction into culture medium supplemented with bacterial cellulose of SL-20 and SL-21 strigolactone mimics was determined. The production of non-catalytic proteins of swolenin type with action to loosen the fibrillar structure of cellulose is amplified on average by 83.5% and 72.6%, respectively, in the presence in the culture medium of the undead mimics, SL-20, 2-(4-methyl-5-oxo-2,5-dihydrofuran-2- yloxy)-6-(4-methylpiperidin-1-yl)-benzo[de]izochi-nolin-1 ,3-dione and SL-21 , respectively, 2-(4-methyl-5-oxo-2,5-dihydro-furan-2-yloxy)-6-(4-benzyl-piperidine-1-yl)- benzo[de]isoquinoline-1 , respectively, 3-dione, in concentration of 10-7M.

[0047] Example 4. An experiment was conducted to evaluate the influence of treatment with the strain TpK20, applied alone or together with SL-20 and SL-21 strigolactone mimics, on the production of tomatoes grown in the field under conditions of irrigation deficit, as well as on the accumulation of bioactive compounds, lycopene and gammaamino-butyric acid in tomato fruits.

[0048] The experiment was conducted in the agricultural years 2022 and 2023, on experimental plots located at Valea Crucii <44°27'45" Latitude and 26°31'35" east longitude). The soil in the experimental plots was a cambic chernozem with the characteristics shown in Table 3

[0049] Tab. 3. Characteristics of cambic chernozem from the Valea-Crucii experimental field.

[0050] In the experimental area of Valea Crucii the average multiannual atmospheric temperature is 10.25°C, and annual precipitation amounts to 571 mm. In terms of temperatures, the 2022 season showed variations, with temperatures significantly higher than normal conditions, with a rainfall deficit of more than 120 mm during the growing season of tomatoes and a pronounced atmospheric drought. The 2023 season was also dry, but with a lower rainfall deficit - 80 mm during the growing season of tomatoes. The soil was fertilized with 200 kg / ha equivalent complex fertilizer NPK 15:15: 15 (Azomure§, Targu-Mure§, Romania) before sowing the cover crop, hairy vetch. Conservative working technologies were applied. The tomato crop was established by transplanting seedlings (cv. Benfica) into mulch of hairy vetches, according to technology adapted for European conditions (Campiglia et al. 2010, European Journal of Agronomy, 33, 94-102). The seedlings used to establish the crop were produced in a warm greenhouse using specific technology. The following experimental variants were carried-out:

[0051] > V1 - mulch-covered soil, untreated

[0052] > V2 - mulch-covered soil, soil treatment with SL-20 2-(4-methyl-5-oxo-2,5- dihydrofuran-2-yloxy)-6-(4-methylpiperidin-1-yl)-benzo[de]isoochi-nolin-1 ,3- dione, equivalent dose 1 ,25 g / ha

[0053] > V3 - mulch-covered soil, soil treatment with SL-21 , 2-(4-methyl-5-oxo-2,5- dihydro-furan-2-yloxy)-6-(4-benzyl-piperidin-1-yl)-benzo[de]isoquinoline-

[0054] 1 ,3-dione, equivalent dose 1 ,25 g / ha

[0055] > V4 - mulch-covered soil, foliar treatment T. pseudokoningii Tpk20 applied foliar at a dose equivalent of 1012spores I ha,

[0056] > V5 - mulch-covered soil, soil treatment SL-20 2-(4-methyl-5-oxo-2,5- dihydrofuran-2-yloxy)-6-(4-methylpiperidin-1-yl)-benzo[de]isoochi-nolin-1 ,3- dione, equivalent dose 1 ,25 g / ha, foliar treatment T. pseudokoningii Tpk20 applied foliar at a dose of 1012 spores / ha,

[0057] > V6 - mulch-covered soil, soil treatment SL-21 , 2-(4-methyl-5-oxo-2,5- dihydro-furan-2-yloxy)-6-(4-benzyl-piperidin-1-yl)-benzo[de]isoquinoline-

[0058] 1 , 3-dione, equivalent dose 1 ,25 g / ha, foliar treatment T. pseudokoningii Tpk20 applied foliar at a dose of 1012 spores / ha,

[0059] > V7 - untreated control, soil not covered with mulch

[0060] The experiment was conducted under deficit irrigation conditions. Compared to the estimated optimum, irrigation through the drip irrigation system was 25% lower. Each experimental treatment had 3 repetitions.

[0061] Strigolactone mimics were applied to the soil by spraying a solution containing 0.5% sodium lignosulfonate as a spraying adjuvant. Foliar treatments were applied with TpK20 suspensions obtained by cornmeal medium cultivation. The cornmeal medium stimulates chlamydospore production (Li et al., 2016. Journal of Zhejiang University- Science B, 17, 619-627). The solutions were applied using an SG20 backpack sprayer (Stihl AG, Waiblingen, Germany), by spraying from 40 cm, with a pressure set at 275 kPa, using a flat jet nozzle with limited drift (TeeJett® flat-fan TT11002 model, Spraying Systems Co., Wheaton, IL, USA). Tomatoes were harvested, and the yield was determined. In the harvested tomato fruits, the following compounds were determined: lycopene (Sadler et al. 1990. Journal of Food Science, 55, 1460-1461) and gamma-aminobutyric acid (Henderson et al. 2000. Amino acid analysis using Zorbax Eclipse-AAA columns - Agilent Note, 1100, 1-10). The results obtained are presented in Table 4.

[0062] Tab. 4. Influence of treatment with strigolactone mimics and TPk20 strain suspension on tomatoes yields under deficient irrigation conditions and on lycopene and gamma-amino-butyric acid accumulation on tomato fruits.

[0063] The above data demonstrate that Trichoderma pseudokoningii Tpk20 strain, when applied foliar at a dose of 1012chlamydospores / ha, together with strigolactone mimics, SL-20, 2-(4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)-6-(4-methyllpiperidin-1-yl)- benzo[de]isoochi-nolin-1, 3-dione and, respectively, SL-21, 2-(4-methyl-5-oxo-2,5- dihydro-furan-2-yloxy)-6-(4-benzyl-piperidin-1 -yl)-benzo[de]isoquinoline-1 ,3-dione, applied as soil treatment, at an equivalent dose of 1.25 g / ha, promote yield on deficit irrigation and determine a significantly increased accumulation of lycopene and gammaamino-butyric acid in fruits, compared to the untreated control, or experimental variants treated with only one of the components, Tpk20 or strigolactone mimics.

Claims

Claims1. Strain of Trichoderma pseudokoningii Tpk20, according to the invention, deposited under DSM number 34838 at Leibniz-lnstitute DSMZ - German Collection of Micro-organisms and Cell Cultures, Braunschweig, characterized in that have an antagonistic effect to phytopathogens growing on plant, promotes plant nutrition by producing phytases that mineralize organic phosphorus and siderophores, biosynthesizes non-catalytic proteins of swolenin type with action to loosen the fibrillar structure of cellulose, increases the efficiency of water use in tomato plants grown in the field and increase yield, activates secondary metabolism in treated tomato plants, promote the accumulation of lycopene and gamma-amino-butyric acid, responds to the strigolactones mimics SL-20, 2-(4-methyl-5-oxo-2,5-dihydrofuran-2- yloxy)-6-(4-methylpiperidin-1-yl)-benzo[de]isoquinoline-1 , 3-dione, and SL-21, 2-(4- methyl-5-oxo-2,5-dihydro-furan-2-yloxy)-6-(4-benzyl-piperidin-1-yl)-benzo[de]isoqui- noline-1, 3-dione, by amplifying the production of phytases, siderophores and non- catalytic proteins of the swolenin type, act complementarily with strigolactone mimics on tomato plants grown in the field, in increasing water use efficiency, promoting yield and accumulation of lycopene and gamma-amino-butyric acid in tomato fruits.

2. Trichoderma pseudokoningii Tpk20 strain according to claim 1 , characterized in that production of siderophores is amplified on average by 45.3 % and, respectively, by 63.8%, by the presence in the culture medium of the strigolactone mimics, SL-20, 2-(4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)-6-(4-methyllpiperidin-1 - yl)-benzo[de]isoochi-nolin-1 ,3-dione and, respectively, SL-21 , 2-(4-methyl-5-oxo-2, 5- dihydro-furan-2-yloxy)-6-(4-benzyl-piperidin-1-yl)-benzo[de]isoqui-noline-1, 3-dione at a concentration of107M.

3. Trichoderma pseudokoningii Tpk20 strain according to claim 1 , characterized in that the production of phosphorus-solubilizing compounds is amplified on average by 72.3% and, respectively, 82.8%, by the presence in the culture medium of strigolactone mimics, SL-20, 2-(4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)-6-(4- methylpiperidin-1-yl)-benzo[de]isoochi-nolin-1, 3-dione, and, respectively, SL-21, 2-(4- methyl-5-oxo-2, 5-dihydro-furan-2-yloxy)-6-(4-benzyl-piperidin-1-yl)-benzo[de]isoqui- noline-1, 3-dione at a concentration of 10'7M.

4. Trichoderma pseudokoningii Tpk20 strain according to claim 1, characterized in that the production of non-catalytic proteins of the swolenin type with action to loosen the fibrillar structure of cellulose is amplified on average by 83.5% ancLrespectively, by 72.6%, in the presence in the culture medium of strigolactone mimics, SL-20, 2-(4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)-6-(4-methylpiperidin-1 - yl)-benzo[de]izochi-nolin-1, 3-dione and, respectively, SL-21 , 2-(4-methyl-5-oxo-2,5- dihydro-furan-2-yloxy)-6-(4-benzyl-piperidin-1-yl)-benzo[de]isoquinoline-1 ,3-dione, at concentrations of 107M.

5. Trichoderma pseudokoningii strain Tpk20 according to claim 1 , characterized in that when applied foliar at a dose of 1012spores / ha, together with strigolactone mimics, SL-20, 2-(4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)-6-(4-methyllpiperidin-1- yl)-benzo[de]isoochi-nolin-1 ,3-dione and, respectively, SL-21 , 2-(4-methyl-5-oxo-2,5- dihydro-furan-2-yloxy)-6-(4-benzyl-piperidin-1 -yl)-benzo[de] isoquinoline- 1 ,3-dione, applied as soil treatment, at an equivalent dose of 1.25 g / ha, promotes yield in deficit irrigation conditions and determines a significantly increased accumulation of lycopene and gamma-amino-butyric acid in tomatoes fruits compared to the untreated control, or experimental variants treated with only one of the components, Tpk20 strain or strigolactone mimics.

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