Inhibitor of andis virus of potato mottling

FIELD: biotechnology.

SUBSTANCE: invention relates to the use of the concentrate of the culture liquid of the strain Trichoderma harzianum Rifai, deposited in the Russian National Collection of Industrial Microorganisms under the number of RNCIM F-180 as an inhibitor of Andis virus of potato mottling.

EFFECT: invention enables to reduce losses of potato from the plant infection with the Andis mottling virus.

2 ex

 

The invention relates to biochemistry and may find application in agricultural biotechnology, Microbiology and plant breeding.

A well-known inhibitor of the herpes virus simple 1-th type (1), a known inhibitor of human immunodeficiency virus (2). As inhibitor was used metabolite strain Trichoderma harzianum Rifai - homogeneous enzyme L-lysine α-oxidase.

In connection with the importation into Russia of a large number of imported material of potato, there is a risk of infection of healthy plants of the Andean potato virus craptastic potatoes. Also a great risk of spreading infected potato associated with an increase in the international exchange of seed and genetic material in the form of tubers and in vitro culture and true seeds (3, 4).

Andean virus craptastic potatoes (Andean potato mottle virus) is widely distributed in the highlands of the Andes in Chile, Ecuador and Peru, and Brazil. It mainly affects the potatoes, however, can be found on the eggplant and some other solanaceous crops (3, 5).

Description inhibitors Andes virus craptastic potatoes (Andean potato mottle virus) not found.

The technical result of the invention is to reduce losses from infection of potato plants in the Andes virus craptastic potatoes.

The technical result is achieved by the fact that the proposed application is concentrate the culture fluid of strain Trichoderma harzianum Rifai, deposited in Russian national collection of industrial microorganisms under the number F-180, as an inhibitor in the Andes virus craptastic potatoes.

The strain is known (6), deposited in Russian national collection of industrial microorganisms under the number VKPM F-180 (Moscow, Road 1-y passage, 1. Institute of genetics and breeding, 1987).

The cultivation conditions of a strain of Trichoderma harzianum Rifai.

Fermentation of the strain was carried out on the equipment Experienced process plant IBPM RAS. Gscreen (Pushchino).

Used the fermenter type BIOR-01 produced by BLS Bureau, city of Kirishi, volume 100 l with a fill factor of 0.6. The fermenter equipped with a magnetic stirrer, filters air, temperature and pH.

Nutrient medium was prepared directly in the apparatus. For this purpose, the apparatus was filled with 60 l of tap water, contributed 1% wheat bran, stimulant-0,1 %, 1.3% of ammonium sulfate, the pH value of 5.8-6.0 installed a 10% solution of Hcl. Wheat bran and stimulant pre-soaked in 10 litres of water for 4 hours, were sterilized in an autoclave for 1 hour at t° +125°C. Then prepared the fermenter was seeded with seed material from the flasks. Sowing dose of at least 5%. Cultivation was carried out at a temperature of +26°C, air flow throughout the fermentation 30 litres per minute, the speed is Esaki 200 rpm. The pH value in the process of growth was adjusted to 6.5. Duration of cultivation was 94-98 hours, the final pH from 5.3 to 7.5.

At the end of the fermentation culture liquid was sent to the site pre-treatment, where the fungal mycelium was separated by filtration under vacuum on the suction filter. The weight of the obtained biomass was 3.5 kg Received native solution was subjected to additional separation in the separator of the type RSD at 9000 rpm for one hour at a temperature of +2 to +4°C. Then the native solution in a volume of 55 l, obtained after separation of the biomass was concentrated to 1.5 l (concentrate).

Ribonucleic acid (RNA) in the Andes virus craptastic potato was extracted from positive control (firm DSMZ, Germany). The samples were ground in a porcelain mortar until a homogeneous state with the addition of lytic buffer from the set of "Sample NC", company OOO Agravante". From pre-prepared samples were isolated RNA using sets of "Standard NC", company OOO Agravante", according to the Protocol from a set.

For the production of reverse transcription (FROM) in a test tube was added the following mixture for FROM: FROM-buffer AMV company Promega (250 mM Tris-HCl, 250 mM KCl, 50 mM MgCl22.5 mM spermidine, 50 mM DTT) and 5 μl of the mixture dNTP company CJSC Dealt LTD (da, dt, DG, DC - 200 µm each), and 2.5 μl, Rnasin - 40 units (1 μl), revertase AMV firm "romega" (10 U/μl) - 3 μl of water to a total volume of 20 μl of the RNA sample was 5 μl. The reverse transcription reaction (the formation of double-stranded DNA by matrix-stranded RNA) occurred at t° +42°C for 1 hour, at t° +95°C - 5 minutes.

At the stage of reverse transcription to isolated and purified RNA virus, Andes virus craptastic potato was added to the concentrate of the culture fluid in different dilutions (from 5 to 0.025 U/ml). Next to amplification reaction was prepared with the following mixture: 10x MagMIX - 2025 closed joint stock company "Dialout-LTD - 2,5 μl of the sample, the primers of the Armu river direct and inverse 2 μl (10 ρmol/μl) to the sample, cDNA and 5 μl of water to 25 μl of mineral oil. As a result, the complimentary deoxyribonucleic acid (cDNA). To obtain the reaction products (amplicons) were heated mixture as follows: one cycle at t° +96°C for 15 minutes, 30 cycles at t° +96°C for 30 seconds, at t° +65°C for 30 seconds and at t° +72°C for 30 seconds; one cycle at t° +72°C for 10 minutes. Storage was carried out at t° +4°C. amplicons (repeatedly increased the number of copies of the study area cDNA) was made in a 1.5% agarose gel, after which he received electrophoregram and evaluate the results.

Example 1.

After carrying out reverse transcription to samples selected cDNA was added to the enzyme at concentrations of 1 U/ml of 2.5 U/ml and 5 U/ml Was also taken control sample cDNA, which was not added enzyme. On electrophoregram the presence of dark bands in the sample testified to the preservation of a virus, no stripes meant his destruction. At a concentration of 1 U/ml or more cDNA of the virus is destroyed, with no formation of the dark stripes.

Example 2.

After carrying out reverse transcription to samples selected cDNA was added to the enzyme at different concentrations (1, 0,1, 0,05, of 0.025 U/ml). Was also taken control cDNA sample without added enzyme. On electrophoregram the presence of dark bands in the sample testified to the preservation of a virus, no stripes meant his destruction. At a concentration of more than 1 U/ml cDNA of the virus was destroyed, this was not observed the formation of the dark stripes. At a higher dilution of the enzyme (0,1, 0,05, of 0.025 U/ml) viral cDNA is saved and there is a dark band.

Sources of information

1. Inhibitor of herpes simple 1-St type. RF patent № 2022012, 1994. S.B. Alekseev, birch CT, Andjaparidze OG and others.

2. Inhibitor of human immunodeficiency virus. RF patent № 2022011, 1994. S.B. Alekseev, Weight B.C., Smirnov I.P., and others.

3. ERRO. Phytosanitary procedures PM 3/21 (2). Post-entry quarantine for potato // Bulletin OEPP/EPPO Bulletin, 2004, vol.34, p.443-454.

4. Fribourg, C.E.; Jones, R.A.C.; Koenig, R. (1979) Andean potato mottle virus. CMI / AAB Descriptons of Plant Viruses No.203. Association of Applied Biologists, Wellesboume, UK.

5. Salazar, L.F.; Harrison, B.D. Particle properties and strains of Andean potato mottle virus. Journal of General Virology, 1978, 39, 171-178.

6. Smirnov I.P., birch CT, Chekunova LN. Strain Trichoderma harzianum Rifai producer L - Lisino-α-oxidase. The copyright certificate. 1991, No. 1044043.

The concentration of the culture fluid of strain Trichoderma harzianum Rifai, deposited in Russian national collection of industrial microorganisms under the number F-180, as an inhibitor in the Andes virus craptastic potatoes.



 

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