LV14804B - Biologically active composition inhibiting botrytis cinerea infection and a method for producing it - Google Patents

Abstract

Invention concerns to forest chemistry branch and covers technology of complete utilization of tree biomass by obtaining biologically active substances with antifungal properties against the fungi Botrytis cinerea. The aim of invention is biologically active products' with antifungal properties against fungi Botrytis cinerea development. The aim is achieved by new product contains spruce bark ethanol extract, ethanol with stabilizer and preservative, sticky agent, emulsifier, potassium hydroxide solution and water.

Description

DESCRIPTION OF THE INVENTION

The invention relates to the field of forestry chemistry and concerns the complex processing of plant-based raw materials to obtain biologically active products with fungicidal properties against gray rot (Botrytis cinerea).

TECHNICAL LEVEL

Gray rot (Botrytis cinerea) is one of the most common fungal diseases that infect various plants, causing significant economic losses: strawberries account for 40% of Botrytis cinerea and 20% for raspberries.

Gray rot (Botrytis cinerea) is spread by conidium spores. Under favorable conditions, spores germinate even within 24 hours. Airborne temperatures (+15 - +23 ° C) and long-term increased humidity (rain, fog, dew) are factors contributing to the spread of gray rot.

Plant protection against Botrytis cinerea is focused on reducing spore formation (sporulation) and germination, as well as inhibiting mycelial growth.

There are data on the fungicidal activity of Viola methanolic extracts of plants against Botrytis cinerea [1]. A mixture of capric acid, ferulic acid, caffeic acid and p-hydroxybenzoic acid has been found to inhibit mycelial growth of Botrytis cinerea [2],

The plant material is based on the pine (Pinus sylvestris L.) and spruce (Picea abies (L.) Karst.) Lichen: needles and non-woody twigs. Plant protection uses conifers extracts or fractions thereof, pine oil, other products, various compounds, terpenoids and phenols, which increase the plant's resistance to various herbivores and microorganisms [3].

There is a well-known remedy «Fitesten» (thick needle of conifers, SBE), which is obtained from coniferous sapwood according to the requirements of Latvian company US 000312820-08-99 or GOST 21769-84. The product is produced in the form of a thick paste. The preparation is registered in the Register of Medicinal Products of the Republic of Latvia under No. 95-0002. Complex needle thick extract (SBE) has a high biological activity because it contains biologically active substances - carotenoids, polyphenols, chlorophyll derivatives, isoabienol. Vitamins K and E, diterpenes and triterpenes, resin acids, fatty acids, sterols, polymeric compounds and the like. It has the properties of a phytoantibiotic, an antioxidant, a biostimulant, a hepatoprotector, a cell membrane protector and an immunomodulator. SBE has a thick consistency, dark green color and a specific needle smell and taste [4, 5].

The composition of a resin complex derived from coniferous extract containing terpene compounds is known: menthol, terpeniol, camphor, bomeol, caran, spinach and bomane. The composition releases arachinic acid and inflammatory mediators and is therefore used to reduce the inflammatory response [6, 7].

Due to the high content of biologically active substances, extracts of coniferous algae and their processed products are widely used in pharmaceuticals, cosmetics, agriculture and household chemistry.

There is a known method for obtaining biologically active substances from extracts of tree lingonberry, which contains a natural resin solution in hydrocarbon solvents. Coniferous green grass is crushed to a destructurized mass with a moisture content of 40-50%, and extraction is performed with a non-polar aliphatic hydrocarbon solvent, such as light petroleum. During the extraction process, the mass is heated to 40-45 ° C with stirring and stirred for 4-4,5 hours, then cooled to 20-25 ° C and held for 20-24 hours. The extracted mass is divided into the liquid phase, the extract containing water-hydrocarbon extract, and the solid phase, the extracted casing. The water-hydrocarbon extract releases the natural resin solution and the aqueous oligosaccharide solution dissolved in the hydrocarbon solvent, while the biologically active substances are released from the solid phase: bioflavonoids and dihydro-quercetin [8,9].

The complex processing of vegetable raw materials yields valuable, biologically active products used in the pharmaceutical, cosmetic, agricultural, household chemical and food industries.

Some chemical plant protection products have been authorized to reduce damage to Botrytis cinerea. The use of chemical plant protection products on organic farms is prohibited. Therefore, it is urgent to develop new, environmentally friendly plant protection products based on vegetable raw materials.

The object of the invention is to develop biologically active products with fungicidal properties against gray rot (Botrytis cinerea).

The objective is achieved by the composition of the new product comprising ethanol extracts of spruce bark, ethanol with a stabilizer and preservative, a glidant, an emulsifier, potassium hydroxide solution and water. In addition, the biologically active composition contains ethanol extract of spruce bark at 30% concentration, 96% ethanol with 1% stabilizer and 0.2% preservative, solids, emulsifier, potassium hydroxide solution and water in the following weight ratios (mg):

Spruce Bark Ethanol Extract with 30% Extract Concentration% Ethanol with 1% Stabilizer and 0.2% Preservative Solvent Emulsifier Potassium Hydroxide Solution Water

190.0-210.0

347.0-387.0

30.0-34.0

22.5-27.5

1.5- 2.5 352.8-392.8.

The biologically active composition is obtained by crushing the bark of the fir tree stem, treating with ethanol, boiling the bark with ethanol, filtering, filtering off the ethanol extract of the bark, collecting and distilling off the ethanol extract, adding ethanol with a stabilizer and preservative, water, lubricants, emulsifiers and potassium hydroxide solution. The bark of the fir tree stem is cut to size up to 4 mm, treated with 96% ethanol in the ratio: bark - 96% ethanol 1: 6-7, bark with 96% ethanol is boiled at 78 ° C for 160-200 min, filtered, filtered bark ethanol extract Allow to stand for 120 minutes, collect and distil the ethanol extract to a concentration of 30% fir bark in ethanol extract, take 96% ethanol, add stabilizer to preservative in the following proportions: 96% ethanol - stabilizer - preservative - 382: 3.8: 0.76, ie 96% ethanol is prepared with 1% stabilizer and 0.2% preservative, then spruce bark ethanol extract is added with 30% extract, water, adhesive, emulsifier and 10% potassium hydroxide solution in the following ratios: 96% ethanol with stabilizer and ethanol extract of spruce bark preservative with 30% extract concentration - water solids - emulsifier - 10% potassium hydroxide solution 1: 1.86: 1.84: 0.16: 0.13: 0.6.

Changes in technological techniques and regimes in the process of obtaining a new composition result in changes in the composition complex.

The qualitative properties of the new biologically active composition can be determined by the content of biologically active spruce bark ethanol soluble compounds in its content.

Qualitative analyzes of the new biologically active composition inhibiting gray rot (Botrytis cinerea) were performed in the laboratory of the Latvian State Forestry Research Institute “Silava” using gas chromatography method ACME (YoungLin Instrument Co, module 6100 with flame ionization detector (FID) and automatic program (Autochro - 2000)

Examples of new composition samples

Examples I - II

The biologically active composition which inhibits gray rot (Botrytis cinerea) contains spruce bark ethanol extract with 30% extract concentration, 96% ethanol with 1% stabilizer and 0.2% preservative, adhesive, emulsifier, 10% potassium hydroxide solution and water in the following components by weight (mg):

Spruce bark ethanol extract with 30% extract 190.0-210.0 concentration 96% ethanol with 1% stabilizer and 0.2% preservative 347.0 - 387.0 adhesive 30.0-34.0 emulsifier 22.5-27.5 potassium hydroxide solution 1.5-2.5 water 352.8-392.8.

Method for obtaining a biologically active composition which inhibits gray rot (Botrytis cinerea) Shreds 3000 g of spruce bark from saw logs up to 4 mm in size. The crushed bark is treated in an extractor with 96% ethanol in the ratio bark to 96% ethanol 1: 6-7. Connect the contents to the condenser and cook at 78 ° C for 160-200 min. Stop cooking and filter the contents. Leave the filtered bark for 120 minutes. and the ethanol mass is collected. The resulting ethanol mass is distilled to a concentration of up to 30% ethanol extract of spruce bark. Add 96% ethanol stabilizer (polysaccharide - xanthan) and preservative (potassium sorbate) ratios: 96% ethanol - stabilizer (polysaccharide - xanthan) preservative (potassium sorbate) - 382: 3.8: 0.76, then spruce bark ethanol extract with 30% extract, water, adhesive (Trifolio S Forte), emulsifier (Polysorbate 80) and 10% potassium hydroxide solution in the following ratios: 96% ethanol with stabilizer (polysaccharide - xanthan) and preservative (potassium sorbate) spruce bark ethanol extract containing 30% extractives - water - solids (Trifolio SForte) - emulsifier (Polysorbate 80) - 10% potassium hydroxide solution 1: 1,86: 1,84: 0,16: 0,13: 0,6.

In the process, Trifolio S Forte, Aventrol, Medalum and the like may be used as the adhesive, and polysorbate 80, Tvveen 80, Glyceryl monostearate, Sodium steaoryl, Tegoamid, etc. may be used as emulsifiers, and the polysaccharide xanthan, trisodium phosphate, ), white acacia resin, Cellulose etc., potassium sorbate, calcium sorbate, sorbic acid, sodium citrate, etc. may be used as preservatives.

The resulting blend of spruce bark ethanol extracts has a biologically active composition with fungicidal properties against gray rot (Botrytis cinerea), as demonstrated by laboratory and field studies.

Laboratory and field studies

In the experiments, the reference strain of phytopathogenic fungus (Botrytis cinerea) obtained from the Latvian Collection of Microorganisms was used to evaluate the fungicidal properties. Laboratory isolates of infected fungi were used for experimental comparison.

(Botrytis cinerea) (Per.) LMWC no. 658, originating in Latvia;

(Botrytis cinerea) LUBI - Zl isolated from strawberries (201 l);

(Botrytis cinerea) LUBI - 4B isolated from raspberries (2010).

During the experiments, the fungi were grown on potato dextrose agar medium. For prolonged storage, the fungi were inoculated on a must agar medium.

Experiment 1

Determination of the effect of the new biologically active composition on the growth of (Botrytis cinerea) mycelium

Various concentrations of ethanol extract of spruce bark were used in the experiments. 10.0 and 20.0 g / L ' ¹ . One of the classical methods for assessing the efficacy of extracts and fungicides was used: fungal growth radial assay based on mycelial growth changes in microscopic fungal test cultures [10],

Potato dextrose agar medium (PDA) was used for cultivation. Ethanol extracts of spruce bark were added to the medium at various concentrations after autoclaving. Control plates (Botrytis cinerea) were grown on PDA medium to which no spruce bark ethanol extracts were added. 5 replicates are prepared for each variant.

At the start of the experiment, a fungal isolate slice (5x5mm) from the control, grown from the culture for 10 days, was placed under sterile conditions in the center of the control and experimental plates. The plates were then placed in an incubation chamber at 23 ± 2 ° C and 80% relative humidity. The fungal colony diameter (2 diagonal measurements) was measured daily until the fungus filled the entire plate. Mycelial growth inhibition coefficient (P) was calculated using the formula:

DC-DT

DC xl00, where

P - inhibition coefficient,

DC - mean colony diameter in control,

DT - mean colony diameter in the test variants.

The effective concentration (EC50) at which the micelle is inhibited by 50% is called the inhibition coefficient [10,11],

The experiment showed that ethanol extract concentrations of spruce bark: 1; 10 and 20 g / L ¹ slowed mycelial growth of all strains of Botrytis cinerea used in the experiments, and colony diameter was significantly different (control 99%, p <0.01) after 72 hours and 120 hours in all variants (Table 1). ).

A mycelial growth inhibition coefficient of greater than 50% indicates the fungicidal activity of the test substances. The results show that after 72 hours in strains and Botrytis cinerea (Per.) LMKKNr. 658 and Botrytis cinerea LUBI - 4A, mycelial inhibition coefficient in all variants with added spruce bark ethanol extracts is greater than 50% (Table 1). In variants with added spruce bark ethanol extracts, the inhibition coefficient is 72.1%, 87.0% and 90.9% after 72 hours. Inhibitory effects were also observed after 120 hours at 94 g, 71.0% and 76.2% with ethanol extract of spruce bark at 20 g / L ' ¹ .

Table 1. Effect of ethanol extracts of spruce bark on growth of mycelial strains of Botrytis cinerea on potato dextrose agar medium, dose dependence:

Used strain Dose added to culture medium, g L ' ¹ Colony diameter, mm Mycelial growth inhibition coefficient (P) (%) after 72 hours after 120 hours after 72 hours after 120 hours Botrytis cinerea LUBI-Z1 0 (control) 63.3 ± 0.5 * 85.00 ± 0.0 * - - 1.0 37.7 ± 1.1 48.9 ± 1.4 40.4 ± 1.7 e 42.4 ± 1.7 d 10.0 19.6 ± 2.2 30.5 ± 5.2 71.6 ± 6.5 c 64.1 ± 5.5 cb 20.0 5.0 ± 0.0 5.0 ± 0.0 92, l ± 0, l a 94, l ± 0.0a Botrytis cinerea LUBI-4B 0 (control) 38.0 ± 2.0 * 77.2 ± 7.1 * - - 1 15.1 ± 1.3 24.5 ± 2.0 60.6 ± 1.3 c 68.2 ± l, lb 10th II, 8 ± 0.3 26.5 ± 1.7 69.3 ± 0.8 c 65.6 ± 1.9 cb 20th 5.0 ± 0.0 22.4 ± 0.2 87.0 ± 0.0 b 71.0 ± 0.3 b Botrytis cinerea LMKKNr. 658 0 (control) 54.0 ± 4.6 * 84.2 ± 1.5 * - - 1 23, l ± 0.8 51.5 ± 4.8 57.2 ± 3.0 cd 37.9 ± 1.9 d 10th 21, l ± 0.9 43.6 ± 0.9 61.0 ± 0.8 c 48.3 ± 1.3 c 20th 5.0 ± 0.0 19.7 ± 2.3 L 90.9 ± 0, l a 76.2 ± 0.6 b

Explanations: * marked control values are significantly different from other variant results (p <0.01); values marked with different letters differ significantly within the same column (p <0.05).

Experiment 2

Effect of the new biologically active composition on Botrytis cinerea sporulation

Spore formation is important for the spread of phytopathogens and for plant infection. When creating fungicides, it is important to know whether the resulting biologically active composition inhibits spore formation. The strain of Botrytis cinerea LUBI - Z1, which is characterized by spore formation, was used in the experiment. Sporulation was assessed using PDA medium with spruce bark reference extracts added. The strain of Botrytis cinerea LUBI - Z1 was grown in the incubator (22 ° C) until the mycelium reached the edge of the Petri dish. When sporulation was observed, 3.0 cm ² of culture medium and mycelium pieces were taken and the spores carefully removed from the mycelium with sterile distilled water. As a result of these operations, a spore suspension was obtained, filtered and volume measured. Spore counting under a light microscope was performed using a Goiyev camera. In each variant, the amount of spores of Botrytis cinerea was calculated and plotted against 1 mm.

The results showed that addition of bark fir ethanol extract at 10 g / L ¹ reduces sporulation in the medium by more than 53% (Table 2). The addition of 20 g / L ' ¹ showed significant differences from the control.

Table 2. Effect of ethanol extracts of spruce bark on sporulation of Botrytis cinerea LUBI - Z1 in a dose-dependent manner

Option Dose added to culture medium, g / L ' ¹ Mean number of spores per 1 mm ² medium ± SD Number of spores against control% Control 0 668.08 ± 95.74 1 0.1 587.64 ± 54.90 87.9 2 1.0 555.41 ± 98.21 83.1 3 10.0 360.51 ± 56.39 * 53.9 4 20.0 N Explanation: No growth of mycelium on culture medium and no sporulation was observed at N - 14 days; * the values recorded differ significantly from control results (p <0.05);

3. Field trials

Evaluation of the effectiveness of ethanol extracts of spruce bark against gray rot under field conditions

Prepared form of ethanol extracts of spruce bark, tested in autumn raspberry planting in 2012 season. Working solution of ethanol extracts of spruce bark in three different concentrations - 1%, 2%, 4% - was used. Treatments were started at the beginning of flowering (BBCH - 61) and continued at an average interval of seven days. A total of 8 sprays were applied. The efficacy of ethanol extracts of spruce bark was compared with the untreated control. The trial evaluated the effect of the preparation on raspberry yield, average weight of 50 berries and weight of damaged berries. Autumn raspberry harvest was tested and evaluated 8 times. Ethanol extracts of spruce bark did not significantly affect the raspberry yield and the weight of 50 raspberries at any of the concentrations used.

Spruce bark ethanol extracts, at the highest concentration of working solution, showed the highest efficiency in controlling gray rot. In the variant using spruce bark ethanol extracts at 4% working solution, the weight of the gray berries damaged from the total berry weight was significantly (p <0.05) lower (13.2%) compared to the control (22.1 %).

INDUSTRIAL USE

The newly developed biologically active composition of spruce bark ethanol extracts has fungicidal properties against gray rot (Botrytis cinerea): it significantly (4 times) slows down the growth of mycelium of all Botrytis Cinerea strains used in the experiments, significantly reduces sporulation (more than 53%), control of gray rot (13% ethanol soluble compounds and 22% control) during field vegetation in the raspberry plantation season.

The new biologically active composition of ethanol extracts of spruce bark did not affect raspberry yield and raspberry berry mass at any concentration used.

The new biologically active composition of ethanol extracts of spruce bark can be widely used in the field of forestry, agriculture and household chemistry.

Sources of information

1. Hammami I., Kamoun N. & Rebai A. (2011). Biocontrol of Botrytis cinerea with essential oil and methanol extract of Viola odorata L. flowers. Archives of Applied Science Research, 3 (5): 44-51.

2. Verovkin, A., Neiberte, B., Shable, I., Rabbit, G., Shulga, G. (2008). Chemical component of bark of the most typical Latvian tree species. Latvian Journal of Chemistry, 2,195-201. (in English).

3. Pat. LV 14570 B, 2013.

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7. Pat. RU 2161482, C2, 2001, A61K 31/10.

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Claims (4)

A biologically active composition which inhibits gray rot (Botrytis cinerea), characterized in that it contains ethanol extracts of fir bark, ethanol with a stabilizer and a preservative, a glidant, an emulsifier, potassium hydroxide solution and water. Biologically active composition according to claim 1, characterized in that it contains ethanol extract of fir bark with 30% extractives concentration, 96% ethanol with 1% stabilizer and 0.2% preservative, glue, emulsifier, 10% potassium hydroxide solution and water in parts by weight (mg): Spruce bark ethanol extract with 30% extract concentration 190.0 - 210.0 96% ethanol with 1% stabilizer and 0.2% preservative 347.0 - 387.0 solids 30.0 - 34.0 emulsifier 22.5 - 27.5 10% potassium hydroxide solution 1,5-2,5 water 352.8 - 392.8. 3. A process for preparing a biologically active composition which inhibits gray rot (Botrytis cinerea), characterized by grinding the bark of a fir tree stem, treating it with ethanol, boiling the ethanol bark, filtering, filtering off the ethanol extract of the bark, collecting the ethanol extract, prepared ethanol with a stabilizer and a preservative, then a finished product containing spruce bark ethanol extract with extracts, water, ethanol with a stabilizer and a preservative, a lubricant, an emulsifier and a potassium hydroxide solution. A process for preparing a biologically active composition according to claim 3, characterized in that the bark of the fir stem is cut to size 4 mm, treated with 96% ethanol: bark - 96% ethanol 1: 6-7, the bark is boiled with 96% ethanol. 78 ° C for 160-200 min., Filter, filter the ethanolic extract bark for 120 min., Collect and distill the ethanol extract to a concentration of 30% extract, prepare 96% ethanol with stabilizer and preservative in the following ratio: 96% ethanol - preservative stabilizer - 382 : 3.8: 0.76 ie 96% ethanol with 1% stabilizer and 0.2% preservative, finished product containing spruce bark ethanol extract with 30% extract, water, 96% ethanol with 1% stabilizer and 0.2% preservatives, glues, emulsifiers and 10% potassium hydroxide solution in the following ratios: Spruce bark ethanol extract with 30% extract concentration - water - 96% ethanol with stabilizer and preservative - glue street emulsifier - 10% potassium hydroxide solution 1: 1.86: 1.84: 0.16: 0.13: 0.6.