WO2015084134A1

WO2015084134A1 - Composition of biologically active substances for inhibition gray rot botrytis cinerea and preparation method thereof

Info

Publication number: WO2015084134A1
Application number: PCT/LV2014/000002
Authority: WO
Inventors: Māris DAUGAVIETIS; Ojārs POLIS; Ausma Marija KORICA; Līga JANKEVICA; Vadims BARTKEVIČS; Līga LEPSE; Regīna RANCĀNE
Original assignee: Latvijas Valsts Mežzinātnes Institūts "Silava"; LATVIJAS UNIVERSITĀTES BIOLOĢIJAS INSTITŪTS, LU aģentūra; Pārtikas Drošības, Dzīvnieku Veselības Un Vides Zinātniskais Institūts "Bior"; Pūres Dārzkopības Pētījumu Centrs, Sia; Latvijas Augu Aizsardzības Pētniecības Centrs, Sia
Priority date: 2013-12-03
Filing date: 2014-02-05
Publication date: 2015-06-11
Also published as: LV14804A; LV14804B

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

COMPOSITION OF BIOLOGICALLY ACTIVE SUBSTANCES FOR INHIBITION GRAY ROT BOTRYTIS CINEREA AND PREPARATION METHOD THEREOF

The given invention pertains to wood chemistry and in particular to biologically active compounds, recovered in complex processing of forest raw material and exhibiting fungicidal effect against gray rot Botrytis cinerea.

Gray rot Botrytis cinerea is one of the most common fungus diseases, which infect a variety of plants, incurring substantial economic loss: e.g. for strawberries the proportion of Botrytis cinerea infected berries may be as high as 40%, for raspberries - 20%.

The Gray rot is spread by Botrytis cinerea fragmentation spores, which in favourable ambient (air temperature +15 - +23° C and increased air moisture over prolonged periods of time due to rain, fog and dew) germinate even within 24 h.

In controlling Botrytis cinerea the focus is on reducing sporulation and spore germination, as well as inhibition of mycelium growth.

Known in the art is the fungicidal effect of Viola plant methanol extracts against Botrytis cinerea [1]. A mix of 7-coumaric acid, ferulic acid, caffic acid, and p- hydrobenzoic acid is known to inhibit the growth of Botrytis cinerea mycelium [2].

Normally, the foliage (needles and non-lignified twigs) of Scots pine Pinus sylvestris L and Norway spruce Picea abies (L.) Karst. is used as forest origin raw material for recovering biologically active substances. Suitable for plant protection are conifer needle extracts or their fractions, pine oil, terpenoides, phenols, and other compounds, which enhance the resistance of plants against the damage by herbivores and microorganisms [3].

Known is also the medicinal preparation Fitesten, resembling a thick needle paste, which is recovered from conifer foliage as specified by the Latvian Enterprise Standard US 000312820-08-99 and GOST 21769-84. The preparation is entered in the Drug Register of the Republic of Latvia under No. 95-0002. The biological activity of the thick needle extract is high as it contains such biologically active substances as carotenoids, polyprenols, chlorophyll derivatives, isoabienol, vilamms K and E, diterpenes and triterpenes, resin and fatty acids, sterols, polymer compounds, etc. The preparation has the properties of phyto-antibiotic, antioxidant, biostimulator, hepatoprotector, cell membrane protector, and imunomodulator. The product is of thick consistence, dark green colour and has specific needle taste and odour [4, 5]. Known in the art is the composition of resinous substance complexes recovered from conifer foliage extract, which contain terpene compounds like menthol, terpineol, camphor, borneol, caran, pinan, and bornane. The compound, emitting arachidonic acid and inflammation mediators, is used for mitigating the reaction of inflammation [6, 7].

Because of high content of biologically active substances the conifer foliage extracts and the products therefrom are widely used in pharmaceutical and cosmetics industries, in agriculture and household chemistry.

Known is a process of recovering biologically active substances from foliar extracts comprising a solution of natural resins in hydrocarbon solvents. The conifer foliage, comminuted to destructurized mass of the moisture content 40 - 50%, undergoes extraction by using non-polar solvent of aliaphatic hydrocarbons, for instance, petroleum ether. During extraction, lasting for about 4 - 4.5h, the mass, while stirred, is warmed up to 40-45° C, then cooled down to 20-25° C and kept for 20 - 24h. Then the liquid fraction of the extract, consisting of hydrocarbon aqueous extract, is separated from the solid foliage residue fraction. Subsequently, from the extract of hydrocarbons extracted is the solution of natural resins and that of oligosaccharides, with such biologically active compounds as bioflavonoides and dihydro quercetine extracted from the solid residue fraction [8, 9].

In complex processing of forest origin raw material recovered are valuable biologically active products for uses in pharmacy, cosmetics, agriculture, household chemistry and food industries.

In registers of chemicals we find some chemical plant protection agents for suppressing Botrytis cinerea. As in biological farming the use of chemicals is out of the question, developing novel, environment-friendly plant protection agents of vegetable origin is a high priority.

SUMMARY OF THE INVENTION

The present invention aim to develop a new biologically active vegetable origin products with fungicidal properties for Gray rot Botrytis cinerea. The aim is achieved by new product comprises the spruce bark ethanol with extractive substances, ethanol with a stabilizer and preservative, sticker, emulsifier, potassium hydroxide solution and water. DETAILED DESCRIPTION OF THE INVENTION

The above described goal has been achieved by entering certain quantities of components into the composition - namely it comprises spruce bark ethanol extract with extractive substances concentration 30%, 96 percent ethanol with stabilizer 1% and preservative 0.2%, sticker, emulsifier, 10 percent potassium hydroxide solution and water in the following ratio of components, mass (mg):

spruce bark ethanol extract with extractive substance 190.0 - 210.0 concentration 30%

96 percent ethanol with stabilizer 1% and preservative 0.2% 347.0 - 387.0 sticker 30.0 - 34.0 emulsifier 22.5 - 27.5

10 percent potassium hydroxide solution 1.5 - 2.5 water 352.8 - 392.8.

Method for preparing the composition comprises the following steps:

a) comminuting spruce stem bark, treating the bark mass by ethanol,

b) boiling the mass of bark with ethanol, filtrating the mass boiled,

c) sedimenting the filtrate of bark ethanol extract with extractive substances, separating out and distilling the ethanol extract with extractive substances, d) preparing the ethanol with stabilizer and preservative,

e) preparing the composition comprising spruce bark ethanol extract with extractive substances, water, ethanol with stabilizer and preservative, sticker, emulsifier, and potassium hydroxide solution.

The above described the following steps has been achieved by entering the certain quantities of components and the concrete regimes realizations of the method actions: a) comminuting spruce stem bark up to the particle size 4 mm, treating the bark mass by 96 percent ethanol in following proportion: bark mass - 96 percent ethanol as 1: 6 - 7,

b) boiling mass of the bark with 96 percent ethanol for 160-200min. at the temperature 78°C, filtrating the mass boiled,

c) sedimenting the filtrate of bark ethanol extract with extractive substances for 120 min., separating out and distilling the ethanol extract with extractive substances to reach the spruce bark ethanol extract with the concentration of extractive substances 30%, 2

4

d) preparing 96 percent ethanol with stabilizer and preservative in following proportion: ethanol - stabilizer - preservative as 382 : 3.8: 0.76, i. e. 96 percent ethanol with stabilizer 1% and preservative 0.2%,

e) preparing the composition comprising the spruce bark ethanol extract with the concentration of extractive substances, 96 percent ethanol with stabilizer and preservative, sticker, emulsifier, 10 percent potassium hydroxide solution, water in following proportion: spruce bark ethanol extract with the concentration of extractive substances 30% - 96 percent ethanol with stabilizer and preservative - sticker - emulsifier - 10 percent potassium hydroxide solution - water as 1 : 1.84:

0.16: 0.13: 0.6 : 1.86.

In recovering foliar products the alterations introduced in the production processes and regimes induce changes in the whole complex of biologically active substances involved.

The qualities of the novel composition of biologically active substances recovered from spruce bark may be described by the biologically active compounds soluble in ethanol, which make up the formulation.

The qualitative and quantitative analyses of the Botrytis cinerea inhibiting composition of biologically active substances were made at the Latvian Forestry Research Institute „Silava", using the gas chromatograph ACME (Young Lin Instrument Co, modulus 6100 with flame ionization detector (FID)) and automatic program (Autochro - 2000).

Examples of the novel composition

Examples 1 and 2

The Botrytis cinerea inhibiting composition of biologically active substances comprises spruce bark ethanol extract with extractive substances concentration 30%, 96 percent ethanol with stabilizer 1% and preservative 0.2%, sticker, emulsifier, 10 percent potassium hydroxide solution and water in the following ratio of components, mass (mg): spruce bark ethanol extract with extractive substance 190.0 210.0 concentration 30%

10 percent potassium hydroxide solution 1.5 - 2.5 water 352.8 - 392.8.

Method of producing the Botrytis cinerea inhibiting composition of biologically active substances

3,000g of spruce sawlog stem bark comminuted to the particle size 4mm is in an extractor treated by 96 percent ethanol following a proportion: ethanol - bark as 1 : 6 - 7. The mass is boiled for 160 - 200 min. at the temperature 78° C, followed by filtration and the filtrate sedimentation for 120 min.; the ethanol extract is separated from the filtrate and distilled to reach the concentration of bark ethanol extractive substances 30%. Stabilizer (polysaccharide xanthan) and a preservative {potassium sorbate) are added to the 96 percent ethanol following a proportion: ethanol - stabilizer - preservative as 382 : 3.8 : 0.76, i.e. 96 percent ethanol with stabilizer 1% and preservative 0.2%. Then added to the mass the spruce bark ethanol extract of the concentration of extractive substances 30%, sticker, emulsifier and 10 percent potassium hydroxide solution and water following a proportion: spruce bark ethanol extract with the concentration of extractive substances 30% - 96 percent ethanol with stabilizer and preservative— sticker - emulsifier - 10 percent potassium hydroxide solution - water as 1 : 1.86 : 1.84 : 0.16 : 0.13 : 0.6.

As a sticker we may use Trifolio S Forte, Aventrol, Medalum, etc. ; as emulsifier - Polisorbate 80, Tween 80, Glyceryl monostearate, Sodium steaoryl, Tegoamid, etc. ; as stabilizer - polysacharides as xanthan, trisodium phosphate, glucono delta-lactone (GDL), resin of bastard acacia, Cellulose, etc.; as preservative - potassium sorbate, calcium sorbate, sorbic acid, sodium citrate, etc.

As proved by the laboratory and field tests, the composition of biologically active compounds recovered from spruce bark ethanol extract by using the method as specified above possesses fungicidal properties suppressing Botrytis cinerea.

Laboratory and field tests

The reference strain of Botrytis cinerea borrowed from the Latvian National Microorganism Collection was used for evaluating in experiments the fungicidal properties of the composition. For comparison used is the isolate separated out in laboratory conditions from infected fungi.

Botrytis cinerea (Pers.) LMKK No 658, Latvian origin;

Botrytis cinerea strain LUBI - Zl, separated from strawberries in 2011;

Botrytis cinera strain LUBI - 4B, separated from raspberries in 2010. During experiment the fungi were cultivated in the agar medium of potato dextrose. For dead storage the fungi were kept on wort agar medium.

Experiment 1

Effect of the novel composition of biologically active substances on Botrytis cinerea mycelium growth

In experiments different concentrations of spruce bark ethanol extract were used - 1.0; 10.0 and 20.0 g/L^"1. One of the conventional methods was used for comparing the efficacy of extractive substances and fungicides, i, e. the radial test of fungi growth following the variations in the mycelium growth of the culture tested [10].

The potato dextrose agar (PDA) was used for cultivation. After autoclave treatment spruce bark ethanol extractive substances of different concentrations were added to the medium. In control plates Botrytis cinerea was cultivated in the PDA with no spruce bark ethanol extract added to the medium. Each variant was made in five replications.

Initially, a tiny lump (5x5mm) of fungi isolate was in sterile environment cut off the control culture, which had been grown for 10 days. Tad plates were placed in the incubation chamber with the ambient temperature 23 ±2° C and relative air moisture 80%. The diameter of the fungi colony was measured daily (two diagonal measurements) till the whole plate was full. The formula below was used to estimate the mycelium growth inhibition coefficient (P):

DC - DT

P= x 100 , where

DC

P - inhibition coefficient

DC - mean diameter of the fungi colony in control

DT - mean diameter of the fungi colony for all test variants.

Inhibition coefficient represents the efficient concentration (EC₅₀) whereat the mycelium growth is inhibited by 50% [10, 11].

As shown by the experiment, spruce bark ethanol extractive substance concentrations 1, 10 and 20 g/L^"1 slowed down the mycelium growth for all the Botrytis cine.re.a strains tested; in all variants the colony diameter after 72 and 120h signilicantly differed from the control (credibility 99 %, p<0,01) (Table 1).

The mycelium growth inhibition coefficient above 50% is an indication of fungicidal effect. As it follows from the experimental results (Table 1), in the experiments with strains Botrytis cinerea (Pers.) LMKK No. 658 and Botrytis cinerea LUBI - 4A, the mycelium growth inliibition coefficient after 72h was above 50% for all the variants, where spruce bark ethanol extractive substances were added to the medium, 92.1%, 87.0% and 90.9%, respectively. The inhibition by adding to the medium the said extractive substances of the concentration 20g/L^_I remained effective also after 120h - 94.1%, 71.0% and 76.2%, respectively.

Table 1. Effect of spruce bark ethanol extractives on the growth of Botrytis cinerea strain mycelium, on a potato dextrose agar medium, depending on dosage

Experiment 2

Effect of the novel composition of biologically active substances on Botrytis cinerea sporulation

Sporulation is essential for the spread of phytopathogens and infestation of plants. In elaborating fungicides it is important to find out whether the respective formulation of biologically active substances inhibits sporulation. Botrytis cinerea LUBI - Zl strain, known for its sporulation, was used in the experiment to evaluate the sporulation on the PDA medium, where spruce bark ethanol extractive substances were added. Botrytis cinerea LUBI - Zl strain was cultivated in the incubator at 22°C till the mycelium reached the rim of Petri dish. As soon as sporulation started, large lumps of the medium and mycelium of the size 3.0 cm² were taken and the spores were cautiously rinsed off by sterile distilled water. The spore suspension obtained was filtrated and the filtrate volume was measured. Spore recording was done in the light microscope, using the Goryaev's chamber. The amount of spores was calculated for each variant, relating it to 1mm² of the medium surface.

As it follows from the test results, adding spruce bark ethanol extractive substances of the concentration 10 g/L^"1 to the medium reduced sporulation by more than 53% (Table 2), while at the concentration 20 g /L^"1 the results significantly differed from the control.

Table 2. Effect of spruce bark ethanol extractives on Botrytis cinerea LUBI - Zl sporulation depending on the dose applied

Field tests

Testing the effect in field conditions of spruce bark ethanol extractive substances on Botrytis cinerea

The preparation of spruce bark ethanol extractive substances was in the 2012 growing season tested on autumn raspberries, using a solution of the said preparation in three different concentrations - 1%, 2%, 4%. The treatment was first applied at the moment when flowering started (BBCH - 61) and continued (totally eight times) on average with a seven-day interval, comparing the results with the control, where no treatment was done. Evaluated were the total yield of berries, the mean mass of 50 berries, and the mass of rot-affected berries. During the test the berries were picked eight times. The spruce bark ethanol extractive substances had for all the concentrations tested no significant effect on the total yield and the mass of 50 berries.

The highest concentration of spruce bark ethanol extractive substances tested appeared to be most efficient for controlling Botrytis cinerea in autumn raspberries. For the variant with spruce bark ethanol extractive substance concentration 4% the mass of rot-affected berries compared to the total was significantly lower (p<0.05), when compared with the control, 13.2% and 22.1%, respectively.

COMMERCIAL PRODUCTION

The proposed composition of biologically active spruce bark ethanol extractive substances, field-tested on raspberries over the growing season, exhibit fungicidal effect against the gray rot Botrytis cinerea, significantly inhibit (4-fold compared with the control) the mycelium growth for all the Botrytis cinerea strains tested, considerably reduce sporulation (by more than 53%), and efficiently control Botrytis cinerea, (with spruce bark ethanol extractive substance concentration 4% in the solution the mass of rot- affected berries was 13.2% against 22.1% for the control).

For all the concentrations tested the said composition of spruce bark ethanol extractive substances used as a fungicide against Botrytis cinerea had no significant effect on the total yield and the mass of berries.

The novel biologically active composition comprising spruce bark ethanol extractive substances may be widely used in wood chemistry, agriculture and household chemistry.

References

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. Verovkins A., Neiberte B., Sable I., Zakis G., Sulga G. (2008). Latvijas raksturlgako koku sugu mizas kimiskais komponentsastavs. Latvijas kimijas zurnals, 2, 195.-201. (in Latvian).

3. Pat. LV 14570 B, 2013.

4. Pat. LV 13566 B, 2007, A61 36/00.

5. Pat. LV 13888 B, 2009, A61K36/15; A61K36/00.

6. Pat. WO 94/28896, 1994, A61K 31/045.

7. Pat. RU 2161482, C2, 2001, A6 IK 31/10. Pat. RU 2252220, CI, 2005, C07D311/40, B01J20/24.

Pat. RU 2165416, CI , 2001, C07D311/40.

Zambonelli, A., Zechini d'Aulerio, A., Bianchi, A. and Albasini, A. (1996). Effects of essential oil on phytopathogenic fungi. Phytopathology, 144: 491-494. Pandey, D.K., Tripathi, N.N., Tripathi, R.D. and Dixit, S.N.Z. (1982). Fungitoxic and phytotoxic properties of essential oil of Hyptis suaveolens. Pfl Krankh Pfl Schutz, 89: 344-349.

Claims

1. Composition of biologically active substances for inhibition Gray rot Botrytis cinerea, characterized in that it comprises the spruce bark ethanol with extractive substances, ethanol with a stabilizer and preservative, sticker, emulsifler, potassium hydroxide solution and water.

2. Composition of biologically active substances according to Claim 1, characterized in that it comprises spruce bark ethanol extract with extractive substances concentration 30%, 96 percent ethanol with stabilizer 1% and preservative 0.2%, sticker, emulsifier, 10 percent potassium hydroxide solution and water in the following ratio of components, mass (mg):

10 percent potassium hydroxide solution 1.5 - 2.5 water 352.8 - 392.8.

3. Method for preparing the composition of the preceding Claim 1 to 2, characterized in that it comprises the following steps:

a) comminuting spruce stem bark, treating the bark mass by ethanol,

b) boiling the mass of bark with ethanol, filtrating the mass boiled,

4. Method for preparing the composition of the preceding Claim 1 to 3, characterized in that it comprises the following steps:

a) comminuting spruce stem bark up to the particle size 4 mm, treating the bark mass by 96 percent ethanol in following proportion: bark mass - 96 percent ethanol as 1 : 6 - 7, b) boiling mass of the bark with 96 percent ethanol for 160-200min. at the temperature 78°C, filtrating the mass boiled,

c) sedimenting the filtrate of bark ethanol extract with extractive substances for 120 min., separating out and distilling the ethanol extract with extractive substances to reach the spruce bark ethanol extract with the concentration of extractive substances 30%,

e) preparing the composition comprising the spruce bark ethanol extract with the concentration of extractive substances, 96 percent ethanol with stabilizer and preservative, sticker, emulsifier, 10 percent potassium hydroxide solution, water in following proportion: spruce bark ethanol extract with the concentration of extractive substances 30% - 96 percent ethanol with stabilizer and preservative - sticker - emulsifier - 10 percent potassium hydroxide solution - water as 1 : 1.84: 0.16: 0.13: 0.6 : 1.86.