RU2701817C1 - Strain stagonospora cirsii g-51 vizr - producer of herbarumine i and stagonolide a - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention relates to the biotechnology. Disclosed is strain of fungus Stagonospora cirsii, which is a producer of herbarumine I and stagonolide A. Strain of fungus is deposited in State collection of microorganisms VIZR numbered G-51 VIZR.

EFFECT: invention provides high production of herbarumine I and stagonolide A.

1 cl, 4 tbl, 4 ex

Description

The invention relates to biotechnology, in particular to the production of biologically active substances and can be used to produce natural phytotoxic compounds: Gerbarumin I and Stagonol A.

Many phytopathogenic fungi are able to form phytotoxic compounds, which are factors of their virulence. Phytotoxins, which have a minimal effect on non-target organisms (warm-blooded, insects, microorganisms, etc.), can be considered as natural herbicides or prototypes of synthetic herbicides (Berestetskiy, 2008 [1], 2017 [2]).

Among fungal phytotoxins, one can especially distinguish gerbarumin I and stagonolide A from the group of 10-membered lactones (Figure 1). Gerbarumin I showed a stronger toxic effect (LD ₅₀ in relation to seedlings of sad shiritsa about 0 05 mM) than the herbicide 2,4-D (Rivero-Cruz et al., 2000 [3]) Stagonolide A significantly inhibited the growth of roots of seedlings with an LD of ₅₀ about 0.005 mM. It was also shown that stagonolide A is phytotoxic for the leaves of a number of weeds as well (Yuzikhin et al., 2007 [4]). Among other phytotoxins from the group of 10-membered lactones, he showed the highest toxicity to the leaves of the Cirsium arvensis and Sow thistle (Berestetskiy et al., 2008 [5]). These substances were recommended as herbicidal compounds of natural origin (Rivero-Cruz et al., 2000 [3]; Yuzikhin et al., 2007 [4]).

The well-known literature producer Gerbarumin I fungus Phoma herbarum TOX-01020 was isolated from the affected leaves of corn (Zea mays) in 1998. The fungus was cultivated on modified M-D-I medium on orbital shakers at a temperature of 28 ° C and an orbital rocking speed of 120 rpm for 15 days. The fungal metabolites were extracted from the culture fluid and mycelium with ethyl acetate. The combined extract (4 g) was fractionated by silica gel column chromatography in a dichloromethane-isopropanol system under an elution gradient mode with an isopropanol content of 0 to 100%. Herbarumin I was isolated from FIV-3 using preparative high performance liquid chromatography (HPLC) with a yield of 0.320 mg / L (Rivero-Cruz et al., 2000 [3])

Stagonol A is produced by the fungus Stagonospora cirsii C-163 (deposited in the VIZR collection as strain VIZR 1.41). The fungus was cultivated for 2 weeks on a modified Chapek medium without stirring, the metabolites were extracted with ethyl acetate. Stagonolide A was detected in the mid-polar fraction when the extract was fractionated on silica gel with a solvent system of hexane-diethyl ether with a yield of 40 mg / l of medium (Yuzikhin et al., 2007 [4]).

For practical use, an in-depth study of gerbarumine I and stagonolide A (for example, studying the mechanisms of action, synthesis of derivatives, determining selectivity), it is necessary to obtain these biologically active compounds in sufficient quantities (from several hundred milligrams to several grams). The disadvantage of the prototype strain P. herbarum TOX-01020 is the extremely low yield of gerbarumin I. The productivity of the strain of the fungus Stagonospora cirsii VIZR 1.41 for stagonolide A is also low. Therefore, the prospects for using these strains as producers of stagonolide A and gerbarumin I were limited.

The objective of the invention is to obtain a universal producer strain capable of forming gerbarumin I and stagonolide A with high productivity exceeding the prototypes of P. herbarum TOX-01020 and Stagonspora cirsii VIZR 1.41, respectively.

The problem was solved by obtaining a highly productive strain of the fungus S. cirsii G-51 VIZR, capable of producing both gerbarumin I and stagonolide A.

Strain G-51 VIZR was selected by comparing the phytotoxic activity of extracts of various strains of S. cirsii of different geographical origin and isolated from different host plants, including one of the prototypes, strain C-163 (table 1). Identification of fungi was carried out according to J.J. Davis (Davis, 1919 [6]).

To obtain extracts, various strains of S. cirsii were cultivated on a modified Chapek medium of the following composition: 45 g / l sucrose, 3 g / l NaNO ₃ , 1 g / l KH ₂ PO ₄ , 0.5 g / l MgSO4, 0.5 g / l KCl, vitamins thiamine - (100 μg) and biotin (5 μg), pH = 6 Media were inoculated with an agar block cut from the edge of a weekly fungal colony on standard potato-glucose agar (Blagoveshchenskaya, 2017 [7]). Cultivation of S. cirsii cultures was carried out in 500 ml conical flasks with 100 ml of medium in 4 replicates. The cultures were incubated for 14 days at a temperature of 24 ° C without shaking.

The liquid extraction of fungal metabolites was carried out with ethyl acetate in 500 ml separatory funnels. 100 ml of culture filtrate and 100 ml of organic solvent were poured into the funnel and vigorously shaken for 1 minute. The lower aqueous phase was discarded, the upper organic phase was filtered through anhydrous sodium sulfate Na ₂ SO ₄ and evaporated to dryness on a rotary evaporator at 40 ° С.

To assess the phytotoxic effect of the extracts, the leaf disc method was used (Berestetskii et al., 2010 [8]). The studied fractions were first dissolved in a small amount of ethanol, then adjusted with water to a predetermined concentration (5 mg / ml for extracts). The final concentration of ethanol in the solution was 5% (by volume), which is non-toxic to plants. From leaves of the middle tier of 3-5-week-old sow thistles, disks 1 cm in diameter were cut with a cork drill. Die cuts were placed in a wet chamber. Each die cut in the center was punctured with a dissecting needle and 10 μl of the test solution was applied to the puncture region. Symptoms (necrosis area) were taken into account after 48 h of incubation at a temperature of 24 ° C and variable artificial lighting (12 h per day).

The maximum phytotoxic activity against leaf disks was demonstrated by the extract from the liquid culture of strain G-51 VIZR. When the extract concentration was 2 mg / ml, the area of necrosis of leaf disks of sow thistle exceeded 90% when using the extract from the culture fluid of strain G-51 VIZR, which was 1.5 times higher than when using the extract from the culture fluid of strain C-163 prototype (table 2 )

¹ share of the affected area in relation to the total area of the leaf disk,%; extract concentration 2 mg / ml

Producer Characteristic

Systematic position. View Stagonospora cirsii J.J. Davis belongs to the Ascomycota division, the Pezizomycotina subdivision, the Dothideomycetes class, the Pleosporomycetidae subclass, the Pleosporales order, the Massarinaceae family

According to the morphology of sporulation, the strain S. cirsii G-51 VIZR was almost identical to the isolate S. cirsii VIZR 1.41 (C-163), the producer of stagonolide A (Yuzikhin et al., 2007 [4]), isolated from diseased leaves of the arthropod (Cirsium arvense ) The strain S. cirsii G-51 has rapid growth on standard agarized nutrient media (potato glucose agar (OCA), oat agar (OA), maltose agar (MA) and Capeca agar with vitamins (NA)) in the dark at 24 ° C . The diameter of the two-week colonies of S. cirsii G-51 on all agar media was over 90 mm. The fungus forms on the KGA colonies of a dark gray color, with a ragged-felt texture. The reverse color is dark gray. On the OA medium, the fungus forms dark gray colonies with small white cotton-like mycelial clusters. On the MA medium, colonies of S. cirsii G-51 VIZR are light gray, felt-velvety. The color of the reverse is from light brown to dark brown. On ChA medium, the fungus forms velvety smoky-gray colonies. The edge of the colonies on all agarized media is uneven, tree-lobed.

The S. cirsii G-51 VIZR strain is stored in test tubes on mowed potato-glucose agar in a domestic refrigerator at a temperature of 5 ° C, as well as at -80 ° C in a 10% sterile glycerol solution.

Example 1. Deep cultivation of a strain of S. cirsii G-51 VIZR

For deep cultivation of S. cirsii G-51 VIZR, Chapek’s modified liquid nutrient medium with vitamins (CAV) was used in the following modification: glucose - 45 g, NaNO ₃ - 3 g, K ₂ NRA ₄ - 1 g, MgSO ₄ × 7H ₂ O - 0.5 g, KCl - 0.5 g, FeSO ₄ × 7H ₂ O - 0.01 g, thiamine - 100 μg, biotin - 5 μg, 10 g of sunflower refined oil as an antifoam, distilled water to 1 l, pH 6 Mushroom cultivation was carried out in a bioreactor with a working volume of 1.5 l from Applikon Biotechnology (Holland) with an ez-Control process control system and BioXpert software. The system was equipped with sensors for pH, temperature, concentration of dissolved oxygen and foam. Fermentation medium (1.4 L) was inoculated with 100 ml of a 7-day-old culture obtained on a surfactant-free medium without oil according to the method described above. Fermentation parameters: air feed rate 1.5 l / min, temperature 24 ° C, the stirring speed of 200 rpm for 2 days and 400 rpm - until the completion of the fermentation process, the pH level was not maintained. Antifoam - vegetable oil (1% of the volume of the medium) was introduced into the medium before sowing the fungus. Cultivated for 7 days, daily taking samples of 30 ml. Prior to analysis, culture fluid samples were stored in a freezer at -20 ° C.

Quantitative analysis of toxins was carried out as follows. Solid-phase extraction of phytotoxins from the filtrate of the culture fluid of the fungus S. cirsii G-51 VIZR and their purification was carried out using a vacuum unit (manifold) and columns with 0.5 g of reversed-phase sorbent Chromabond C 18c (tank volume 3 ml, Macherey-Nagel, Germany) . The sorbent was conditioned by sequentially adding 3 ml of methanol and 3 ml of water to the column (without drying out). 200 μl of culture fluid filtrate was added to the column. The sorbent was washed with 3 ml of water and 3 ml of 25% methanol, then dried with air for 1 minute. Analytes were eluted from the column with methanol (2 ml), the eluate was thoroughly mixed and stored in a freezer at -20 ° C. For the quantitative analysis of phytotoxins, 200 μl of the eluate were taken from the obtained samples, 300 μl of water was added to it and thoroughly mixed. Phytotoxin analysis was performed using an Acquity UPLC BEN C 18 × 50 × 2.1 mm column. The methanol-water system was used as the mobile phase in a ratio of 50:50 (by volume), the flow rate of the mobile phase was 250 μl / min, and the column thermostat temperature was 40 ° С. The volume of the injected sample is 5 μl. The retention time (t _R ) of stagonolide A was 1.4 min, and gerbarumin I was 1.9 min. The error t _R did not exceed 2%. Calibration characteristics expressing the dependence of peak areas on the concentration of stagonolide A and gerbarumin I in the samples were established by absolute calibration for five concentrations of substances in the range from 0.125 to 20 ng in the analyzed sample, corresponding to phytotoxins from 0.625 to 100 mg / l. The experiment was repeated three times, the data are typical of them.

Stagonolide A was detected in the culture fluid on the 4th day of fermentation. Its maximum concentration (116.7 mg / l) was detected on the 5th day of cultivation, then its content in the culture fluid quickly decreased to 24 mg / ml. On the 7th day of fermentation, stagonolide A was not found in the culture fluid. Gerbarumin was detected in 6-day and 7-day culture fluids (38 mg / l and 116.5 mg / l, respectively) (table 3).

The maximum content of stagonolide A (116.7 mg / l) in a 5-day-old culture of the strain S. cirsii G-51 VIZR is more than 2 times higher than the yield of phytotoxin from the culture of the prototype S. cirsii VIZR 1.41 (C-163). The maximum content of gerbarumin I (116.5 mg / l) in a 7-day-old culture of S. cirsii strain G-51 VIZR is more than 350 times higher than the productivity of the prototype P. herbarum TOX-01020.

Example 2. Obtaining stagonolide A and gerbarumin I using deep cultivation of S. cirsii G-51 VIZR

To verify the data on the phytotoxin content in the S. cirsii G-51 VIZR culture filtrate obtained by quantitative analysis using HPLC (Table 3), a preparative isolation of stagonolide A and Gerbarumin I from the culture filtrate was carried out using standard liquid-liquid extraction and column chromatography methods (Sterner, 2012 [9]).

The cultivation of S. cirsii G-51 VIZR in the bioreactor was stopped on the 6th day of fermentation under the conditions described in Example 1. After separation of the biomass, the extraction of metabolites from the filtrate of the culture fluid was carried out with methylene chloride. Coarse separation of the extract (weight about 500 mg) was carried out in a glass column (length 40 cm, diameter 2.5 cm) on silica gel (50 g, particle diameter 70-200 μm, Merck, Germany) in a stepwise gradient: hexane (eluent 1), hexane ethyl acetate (8: 2 by volume, eluent 2), hexane-ethyl acetate (6: 4, eluent 3), hexane-ethyl acetate (4: 6, eluent 4), ethyl acetate (eluent 5), ethyl acetate-methanol (8: 2 , eluent 6), all 200 ml each (4 portions 50 ml each). The preliminary composition of the fractions was carried out using TLC (Silica gel plates 60 F ₂₅₄ , Merck, Germany) in an 8: 2 hexane-ethyl acetate system. The substances were visualized in UV light (254 nm) and the plates were treated with a reagent based on sulfuric acid and anisaldehyde, followed by heating at 105 ° C. Stagonolide A is well visualized at 254 nm and stained with a brown reagent (R _f 0.35). Gerbarumin I at 254 nm is not detected, the reagent is colored blue (R _f 0.15). According to TLC and HPLC / DMD, fraction 3 (50 mg) contained predominantly stagonolide A, fraction 5 (150 mg) contained gerbarumin I with impurities.

, Швейцария) с УФ/ВИД-детектором Образец фракции 5 адсорбировали на 2 г ОФ сорбента (Silica gel 60 silanazied, диаметр частиц 63-200 мкм, Merck, Германия) и вносили в предколонку для сухой загрузки объемом 6 мл. Элюирование метаболитов осуществляли при комнатной температуре с использованием системы растворителей 0.1%-ная муравьиная кислота - ацетонитрил: 15% ацетонитрила в течение 1 мин, градиент от 15 до 100% ацетонитрила в течение 11 мин, ацетонитрил в течение 4 мин. Скорость потока элюента 24 мл/мин, детекция на длине волны 200 нм. Время удерживания (t_R) гербарумина I составило 4.5 мин.Further purification of gerbarumin I was carried out on a Puriflash PF-15C18HP / 20G reverse phase (RP) column (20 g, sorbent particle diameter 15 μm, Interchim, France) using a Sepacore medium pressure preparative chromatography system (

, Switzerland) with a UV / VID detector. Sample of fraction 5 was adsorbed onto 2 g of RP sorbent (Silica gel 60 silanazied, particle diameter 63-200 μm, Merck, Germany) and introduced into the dry-loading pre-column with a volume of 6 ml. The metabolites were eluted at room temperature using a solvent system of 0.1% formic acid - acetonitrile: 15% acetonitrile for 1 min, a gradient of 15 to 100% acetonitrile for 11 min, acetonitrile for 4 min. The flow rate of the eluent 24 ml / min, detection at a wavelength of 200 nm. The retention time (t _R ) of gerbarumine I was 4.5 minutes.

The final purification of phytotoxins was carried out using preparative HPLC on an XBridge Prep C18 250 × 19 mm column with a particle diameter of 5 μm (Waters, USA). The particle chemistry of this column was similar to the sorbent of the Acquity UPLC BEN C18 analytical column. Chromatography was performed at room temperature and the eluent feed rate (water -acetonitrile 30:70) 24 ml / min. The volume of injected sample is 1 ml (approximately 50 mg of a dry sample was dissolved in 300 μl of acetonitrile and 700 μl of water was added). Metabolites were detected at wavelengths of the UV detector 200 and 234 nm. Under these conditions, t _{R of} gerbarumin I - 9 min, t _{R of} stagonolide A - 12 min. The control of the content of these phytotoxins in the chromatographic fractions was carried out as described above by HPLC / DMD.

The output of stagonolide A was about 30 mg / l, gerbarumin I - 50 mg / l, which corresponds to the data shown in table I for a 6-day culture of S. cirsii G-51 VIZR.

Example 3. Obtaining gerbarumin I using solid-phase cultivation of S. cirsii G-51 VIZR

Autoclaved millet groats were used as a solid substrate. To prepare the substrate, 150 g of groats were placed in 1500 ml microbiological mattresses and 100 ml of deionized water was added. The substrate was autoclaved at 121 ° C for 15 minutes. Two-week-old colonies of S. cirsii G-51 VIZR obtained on potato-glucose agar (KGA) were used as seed for solid-phase cultivation. The strain was cultured for 3 weeks at room conditions. Mattresses with a growing solid-phase culture of S. cirsii G-51 VIZR were shaken every 3 days to prevent clumping of the substrate

The solid substrate colonized by the fungus was dried in a laminar box with a stream of sterile air. The resulting material was crushed and extracted twice with 50% aqueous acetone (by volume) for 10 minutes in an ultrasonic bath. The obtained extract was filtered through a paper filter, acetone was distilled off using a rotary evaporator at a temperature of a water bath of 40 ° C. Metabolites of S. cirsii G-51 VIZR were re-extracted from the aqueous extract remaining after evaporation of acetone with methylene chloride. The methylene chloride extract was dehydrated with anhydrous Na ₂ SO ₄ , after which the solvent was distilled off using a rotary evaporator at a temperature of a water bath of 40 ° С. The dry residue was weighed, the mass of the chloromethylene extract was 3.2 g.

Next, the primary separation of the extract was carried out using column chromatography on silica gel Silica gel 60 (Merck, Germany) with a particle size of 63-200 μm. Elution was carried out according to the method of stepwise gradient systems with an ethyl acetate content of from 0 to 100% (by volume). Based on the results of the separation, 7 fractions XM_A - XM_G were obtained. The eluates were evaporated using a rotary vacuum evaporator at a temperature of a water bath of 40 ° C. Herbarumin I (672 mg) was obtained by separation of the XM_B fraction (1 g) by preparative HPLC on a C18 XBridge Prep C 18 19 × 250 mm column (Waters, USA) as described in the previous example.

The yield of gerbarumin I was 462.4 mg / kg of substrate. It is not possible to compare the yield obtained with the productivity of the prototype strain P. herbarum TOX-01020, since there was no solid-phase cultivation of this strain. Stagonolide A was not found in the culture of the strain S. cirsii G-51 VIZR with this cultivation method.

Example 4. Identification and spectrum of biological activity of stagonolide A and gerbarumin I

HPLC-MS / MS and proton and carbon NMR spectra were used to confirm the structure of the isolated compounds. To obtain mass spectra, we used the TSQ Quantum Access ™ chromatographic system (Thermo Scientific, USA) equipped with diode-matrix fast-scanning and mass spectrometric (triple quadrupole) detectors. Chromatography conditions. Zorbax CB-C18 column (Agilent Tech., USA), grain size 1.8 μm, column dimensions 2.1 × 150 mm, gradient 10-100% acetonitrile in 0.1% formic acid, eluent flow rate 250 μl / min, column temperature 35 ° C, the scanning range of the diode array detector is 200-800 nm, the ionization type is HESI, the scanning range is 50-1000 m / z. Methanol was used to dissolve samples of substances. NMR spectra were recorded using an Avance III Ultra-Shield Plus instrument (Bruker, Germany). Deuterated chloroform was used to dissolve samples of substances. Proton spectra were recorded at a frequency of 400 MHz, carbon spectra were recorded at 100 MHz.

In the mass spectrum of the stagonolide A sample we obtained, there were peaks m / z 227.11 [M + H, 100%] ⁺ and 209.12 [M + H-H ₂ O, 25%] ⁺ , in the mass spectrum of gerbarumin I - m / z 229.11 [M + H, 10%] ⁺ and 211.12 [M + H-H ₂ O, 100%] ⁺ , 193.15 [M + H-2H ₂ O, 25%] ⁺ . Mass spectra demonstrate the presence of one hydroxyl group in stagonolide A and two in herbarumine I. The proton and carbon NMR spectra of stagonolide A and gerbarumin I were identical to those published (Rivero-Cruz et al., 2000 [3]; Yuzikhin et al., 2007 [ four]). The obtained DEPT carbon spectra of both substances contained signals of five CH ₂ groups (3 signals of the lactone ring and 2 signals of the n-propyl group).

To assess the phytotoxic effect of extracts and individual compounds, the method of chipped leaf disks of field sow thistle was used (Berestetskii et al., 2010 [8]). The analyzed sample was first dissolved in a small amount of ethanol, then adjusted with water to a concentration of 5 mg / ml. The final concentration of ethanol in the solution was 5% (v / v). From leaves of the middle tier of 3-5-week-old sow thistles, disks of 1 cm in diameter were cut with a cork drill. The disks were placed in a humid chamber, punctured in the center with a dissecting needle, and 10 μl of solution was applied to the puncture region. The diameter of necrosis on leaf discs of sow thistle was taken into account after 48 h of incubation at a temperature of 24 ° C and variable artificial lighting (12 h per day). Stagonolide A and gerbarumin I were found to be approximately equally toxic to field thistle and field thistle. Gerbarumin I showed higher phytotoxic activity against wheatgrass creeping than stagonolid A on the 5th day after treatment (table 4)

Data are presented as mean ± standard deviation

Thus, the strain of fungus S. cirsii G-51 VIZR was proposed as a producer of gerbarumin I and stagonol A. The productivity of the strain allows one to obtain gerbarumin I in the amount of 116 mg / l when cultivated for 7 days on a liquid nutrient medium in a fermenter, which 150 times the productivity of the prototype strain Phoma herbarum TOX-01020. In addition, S. cirsii G-51 VIZR forms gerbarumin I when cultured on solid nutrient medium with a yield of 460 mg / kg of substrate. When deep cultured for 5 days, this fungus also forms stagonolide A with a yield of more than 100 mg / l, which is more than 2 times the yield of this substance when using the prototype strain S. cirsii VIZR 1.41 (C-163)

Literature

1. Berestetskiy A.O. Mushroom phytotoxins: from basic research to practical use (Review) // Applied Biochemistry and Microbiology. 2008.Vol. 44, No. 5, p. 501-514.

2. Berestetsky A.O. Prospects for the development of biological and biorational herbicides // Bulletin of Plant Protection 2017. T. 91, N 1. P. 5-12.

3. Rivero-Cruz J.F., Garcia-Aguirre G., Cerda-Garcia-Rojas C.M., Mata R. Conformational Behavior and Absolute Stereostructure of Two Phytotoxic Nonenolides from the Fungus Phoma herbarum // Tetrahedron. - 2000. - V. 56. - P. 5337-5344.

4 Yuzikhin O., Mitina G., and Berestetskiy A. Herbicidal potential of stagonolide, a new phytotoxic nonenolide from Stagonospora cirsii // J. Agric. Food Chem. 2007. Vol. 55. N 19. 7707-7711.

5. Berestetskiy A., Dmitriev A., Mitina G., Lisker I., Andolfi A., Evidente A. Nonenolides and cytochalasins with phytotoxic activity against Cirsium arvense and Sonchus arvensis: A structure-activity relationships study // Phytochemistry 69 (2008 ) 953-960.

6 Davis J.J. 1919. Notes on parasitic fungi in Wisconsin. V. Transactions of the Wisconsin Academy of Science. 19 (2): 690-704.

7 Blagoveshchenskaya E.Yu. Mycological studies: the basics of laboratory technology. M.: Lenand, 2017.96 s.

8 Berestetsky, A.O., Yuzikhin O.S., Katkova A.S., Dobrodumov A.V., Sivogrivov D.E., Colombet L.V. Isolation, identification and characterization of phytotoxin formed by Alternaria cirsinoxia fungus // Applied Biochemistry and Microbiology. - 2010. - T. 41, No. 1. - S. 84-88.

9. Sterner O. Isolation of microbial natural products. In: Natural Products Isolation. Sarker S.D., Nahar L. (Eds.) Methods in Molecular Biology, vol. 864, Springer Science + Business Media, LLC. 2012. P. 393-413.

Claims (1)

The strain of the fungus Stagonospora cirsii G-51 VIZR - producer of Gerbarumin I and Stagonolide A.