RU2481392C2 - Producer of impatiens necrotic spot virus inhibitor - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry and use of a Trichoderma harzianum Rifai strain as a producer of an Impatiens necrotic spot tospovirus inhibitor. The Trichoderma harzianum Rifai strain, which is deposited in the Russian National Collection of Industrial Microorganisms under No.F-180, is a producer of the homogeneous enzyme L-lysine-a-oxidase, which exhibits marked antiviral activity with respect to Impatiens necrotic spot tospovirus.

EFFECT: reduced loss of decorative and vegetable crops caused by Impatiens necrotic spot tospovirus.

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Description

The invention relates to biochemistry and may find application in agrobiotechnology, microbiology and crop production.

In recent years, hundreds of thousands of decorative flowers have been imported into the Russian Federation from European countries every year, which may be the masters of balsam necrotic spotted tospovirus (Impatiens necrotic spot tospovirus). Due to the fact that this virus has an extremely wide range of host plants on most ornamental and vegetable crops, there is a danger of infection of potential host plants with this virus. The balsam necrotic spotting virus causes significant losses to the production of ornamental crops in many European countries.

Impatiens necrotic spot tospovirus caused great harm to various types of ornamental and vegetable crops grown in greenhouses in Mexico, the United States and southern Canada and Europe. Crop losses reached 100% (4, 5).

The type 1 herpes simplex virus inhibitor (1) and the human immunodeficiency virus inhibitor (2) are known in the art, and the metabolite of the Trichoderma harzianum Rifai strain, a homogeneous enzyme L-lysine-α-oxidase, was used as an inhibitor.

Also known from the prior art is the possibility of using a gene that inhibits balsamic spotting virus by introducing it into a plant of the Balsamic family to obtain transgenic plants resistant to such a virus (US 6528703 B1, 03/04/2003) - prototype. However, this technique is time-consuming and requires large financial and time costs.

The technical result of the invention is to reduce losses of ornamental and vegetable crops caused by the Impatiens necrotic spot tospovirus virus.

The technical result is achieved by the fact that the Trichoderma harzianum Rifai strain is a producer of the balsam necrotic spotting inhibitor tospovirus. The strain was deposited in the All-Russian collection of industrial microorganisms under the number F-180 (Moscow, Dorozhny 1-y passage, 1, Institute of Genetics and Selection, 1987).

Morphological and biochemical characteristics of the strain

The strain Trichoderma harzianum Rifai on wort-agar medium forms fast-growing colonies. The fungal mycelium is colorless, septate, prostrate. On the 4th - 5th day of growth, tufts with conidiophores appear, pillow-shaped, initially white, with a yellow or dark green color over time. Bottle phialides / 9-12µ /, located in whorls of 3 or more. Conidia glued into the head form on each phialid. The conidia of the fungus are round, smooth, small / 3-5µ /, in transmitted light pale green, and in the mass - dark.

On Chapek’s agar medium, the colonies of the producer strain are rapidly growing, but slightly spore-bearing. The strain grows well on the Chapek medium, but the best growth is observed on the wort-agar medium. Agar does not dilute gelatin, milk does not peptone. Aerobic mushroom. Temperature optimum +28 - + 29 ° С. On potato-dextrose agar there is a decrease in growth intensity, the formation of aerial mycelium and sporulation. Optimum conditions for the growth of the fungus at pH 4-6, however, can grow at pH from 1.5 to 9.

Trichoderma harziatum Rifai equally assimilates both ammonium and nitrate salts. The best source of nitrogen from organic compounds is peptone. It grows well on media with asparagine and with glutamic acid. The best sources of carbon are xylose, glucose, sucrose, lactose, galactose, mannitol, and starch. On media with these sugars, intense growth is observed. Alcohols are poorly absorbed methyl, ethyl, dulcite. It assimilates wheat bran well as a carbon source. The inoculum of the fungus grown on a medium with wheat bran gives a positive result when used in fermentation on media of different compositions.

The strain Trichoderma harzianum Rifai has L-lysine-α-oxidase activity. The activity of L-lysine-α-oxidase in the culture fluid Trichoderma harzianum Rifai was calculated by the growth of N ₂ ABOUT ₂ , the amount of which was determined by spectrophotometric orthodiazidine micromethod. The essence of the method is the interaction of all formed in the reaction H ₂ O ₂  with O-dianisidine hydrochloride. The incubation mixture contained 20 μg of peroxidase, 250 μg of O-dianisidine hydrochloride and 0.1-0.5 mg of protein in 1 ml of the final volume. After 20 minutes of incubation in a thermostat at a temperature of + 37 ° С, the samples were cooled to + 4 ° С. The optical density of the colored solutions of the experimental and control (without substrate) samples was measured on an SF-16 spectrophotometer at 540 nm against the second control sample (without peroxidase).

To build a calibration curve, the molarity of freshly prepared solution H ₂ O ₂  determined by permanganometry. L and DL forms of amino acids served as substrates (Reanal, Hungary). Used 0.05 M phosphate buffer. Reanal peroxidase was used as a peroxidase reaction catalyst, and O-dianisidine hydrochloride (Merck, Germany) was used as a proton donor. The unit of activity was taken as the amount of enzyme catalyzing the formation of 1 μmol N ₂ ABOUT ₂  for 1 min at a temperature of + 37 ° C.

The specific activity of the enzyme was expressed by the number of units of activity per 1 mg of protein or 1 ml of culture fluid. Protein was determined by the Lowry method. A 0.05% solution of crystalline bovine albumin (Reanal, Hungary) was used as a standard.

To assess the purity of the drug, we used electrophoresis in 10% PAAG in the presence of sodium dodecyl sulfate, as well as capillary electrophoresis on the Drops 105 device manufactured by Lumex (St. Petersburg).

The cultivation conditions of the strain Trichoderma harzianum Rifai

Fermentation of the strain was carried out on the equipment of the Experimental technological installation of IBPM RAS named after G.K.Skryabin (Pushchino city).

Used a BIOR-01 type fermenter manufactured by OKB TBM, Kirishi, with a volume of 100 l with a fill factor of 0.6. The fermenter is equipped with a magnetic stirrer, fine filters, air, temperature and pH sensors.

The nutrient medium was prepared directly in the apparatus. To do this, 60 l of tap water was poured into the apparatus, 1% of wheat bran was introduced, a stimulant - 0.1%, 1.3% ammonium sulfate, the pH value of 5.8-6.0 was set with a 10% HCl solution. Wheat bran and the stimulator were pre-soaked in 10 l of water for 4 hours, sterilized in an autoclave for 1 h at a temperature of + 125 ° C. Then the prepared fermenter was seeded with seed from flasks. Sowing dose of at least 5%. The cultivation was carried out at a temperature of + 26 ° C, air flow throughout the fermentation 30 l per minute, the speed of rotation of the mixer 200 rpm. The pH during the growth process was adjusted to 6.5. The duration of cultivation was 94-98 h, the final pH values from 5.3 to 7.5.

At the end of the fermentation, the culture fluid was sent to the pre-treatment section, where the mycelium of the fungus was separated by filtration under vacuum on a suction filter. The weight of the obtained biomass was 3.5 kg. The resulting native solution was subjected to additional separation on an OSB separator at 9000 rpm for an hour at a temperature of +2 - + 4 ° С. Then the native solution in a volume of 55 l, obtained after separation of the biomass, was concentrated to 1.5 l (concentrate).

Ribosucleic acid (RNA) of tospovirus necrotic spotting of balsam was isolated from a decorative potted plant Streptocarpus (lat. Streptocarpus) of the Gesneriaceae family. Produced freezing at a temperature of -20 ° C of plant samples (leaves of Streptocarpus ornamental culture). Then, the samples were ground in a porcelain mortar to a homogeneous state with the addition of a lysis buffer from the Sample NK kit of the Agrodiagnostics LLC. RNA was isolated from pre-prepared samples using the Proba NK kits of Agrodiagnostika LLC according to the protocol from the kit.

For reverse transcription (RT), the following RT mixture was added to the tubes: RT buffer AMV from Promega (250 mM Tris-HCl, 250 mM KCl, 50 mM MgCl ₂ , 2.5 mM spermidine, 50 mM DTT) - 5 μ1, a mixture of DNTF manufactured by ZAO Dialat LTD (dA, dT, dG, dTs - 200 μM each) - 2.5 μl, RNazine - 40 units. (1 µ1), Promega AMV revertase (10 U / µl) - 3 µl, water to a total volume of 20 µl of sample RNA - 5 µl. The reverse transcription reaction (the process of the formation of double-stranded DNA on a single-stranded RNA matrix) occurred at a temperature of + 42 ° C for 1 h, at a temperature of + 95 ° C - 5 min.

At the stage of reverse transcription, a concentrated culture fluid in various dilutions (from 5 to 0.025 U / ml) was added to the isolated and purified RNA of the balsam necrotic spot virus. Next, the following mixture was prepared for the amplification reaction: 10x MagMIX - 2025 from Dialat-LTD CJSC - 2.5 μl per sample, INSV primers direct (S1) and reverse (S2) primers - 2 μl (10 ρmol / μl) per sample , cDNA - 5 µl, water up to 25 µ1, mineral oil. As a result, complementary deoxyribonucleic acid (cDNA) was isolated. To obtain reaction products (amplicons), the mixture was heated according to the following scheme: one cycle at a temperature of + 94 ° C for 15 minutes, 30 cycles at a temperature of + 94 ° C for 1 minute, at a temperature of + 48 ° C for 1 minute, and at temperature + 72 ° С for 1 minute; one cycle at a temperature of + 72 ° C for 10 minutes. Storage was carried out at a temperature of + 4 ° C. The resulting amplicons (a multiple increase in the number of copies of the studied cDNA region) were introduced into a 1.5% agarose gel, after which electrophoregrams were obtained and the results obtained were evaluated (6).

Example 1

After reverse transcription, the enzyme was added to the samples of the isolated cDNA at concentrations of 2.5 Units / ml and 5 Units / ml. A control cDNA sample was also taken in which no enzyme was added. On the electrophoregram, the presence of a dark band in the control sample indicated the preservation of the virus, the absence of the band meant its destruction. At a concentration of 2.5 U / ml or more, the cDNA of the virus is destroyed, and no dark band is observed.

Example 2

After reverse transcription, the enzyme in different concentrations (1, 0.1, 0.05, 0.025 U / ml) was added to the samples of the isolated cDNA. A cDNA control sample was also taken without enzyme addition. On the electrophoregram, the presence of a dark band in the control sample indicated the preservation of the virus, the absence of the band meant its destruction. At a concentration of more than 1 U / ml, the cDNA of the virus was destroyed, and no dark band was observed. With greater dilution of the enzyme, viral cDNA is retained and a dark band is observed.

Literature

1. Inhibitor of herpes simplex virus type 1. RF patent №2022012, 1994. Alekseev S.B., Berezov T.T., Andzhaparidze O.G. and etc.

2. Inhibitor of human immunodeficiency virus. RF patent №2022011, 1994. Alekseev SB, Vesa B.C., Smirnova I.P. and etc.

3. Smirnova I.P., Alekseev S.B. Biosynthesis of the antitumor enzyme L-lysine-α-oxidase Trichoderma sp. // Antibiotics and chemotherapy. - 2009 - T. 54. - B.5-6. - S.8-11.

4. OERR / EPRO (2004). Diagnostic protocol for regulated pests. PM 7/34 (1). Tomato spotted wilt tospovirus, Impatiens necrotic spot tospovirus and Watermellon silver mottle tospovirus. Bulletin OEPP / EPRO Bulletin 34, 271-279.

5. Windham A.S., Hale F., Yanes J. Impatiens necrotic spot virus: a serious pathogen of floral crops (1998). Agricultural Extension Service University of Tennessee. SP370A.

6. Mumford R.A., Barker I., Wood K.R. (1996b) An improved method for the detection of Tospoviruses using the polymerase chain reaction. Journal of Virological Methods 57, 109-115.

Claims (1)

The use of the Trichoderma harzianum Rifai strain deposited in VKPM under No. F-180 as a producer of an inhibitor of the balsam necrotic spotting virus.