RU2827161C1 - Composition with antifungal activity - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: composition with antifungal activity relates to biotechnology, microbiology and can be used in agriculture. Composition contains Bacillus subtilis VKM B3701D with titre 1⋅10⁹ CFU/g and 0.2 M CaSO₄⋅2H₂O.

EFFECT: declared composition has a higher degree of growth inhibition by 15,7% in relation to the fungal necrotrophic plant pathogen Alternaria brassicicola VKM F-1864 and by 14,7% in relation to Aspergillus unguis VKM F-1754 compared to a pure culture of Bacillus subtilis VKM B3701D.

1 cl, 3 dwg, 3 ex

Description

The invention relates to the field of biotechnology, microbiology and can be used in agriculture as a basis for fungicides of biological origin in plant growing.

There are known strains of bacteria of the genus Bacillus that have the ability to suppress the growth of phytopathogenic fungi and stimulate the formation of agricultural crop yields.

Patent RU 2808722 (published 04.12.2023) presents an invention describing a biological preparation based on the bacteria Bacillus amyloliquefaciens VKPM B-13788 for protecting vegetable plants from fungal and bacterial diseases. The specified strain is characterized by a broad spectrum of fungicidal activity and suppresses the development of the following phytopathogenic fungi: Alternaria tenuis, Aspergillus niger, Ascoshita cucumis, Fusarium graminearum, Microdochium nivale (Fusarium nivale), Fusarium solani, Fusarium culmorum, Fusarium sporotrichioides, Fusarium avenaceum, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia sclerotiorum, Phoma solanicola, Phomopsis helianthi, Botrytis cinerea, Magnaporthe grisea, Phytophtora infestans.

A biological product for protecting vegetable plants from diseases caused by phytopathogenic fungi and bacteria is a mixture of the culture fluid of the Bacillus amyloliquefaciens strain VKPM B-13788 with a titer of 2-4⋅10 ⁹ CFU/ml and target additives, immobilized on finely dispersed diatomite granules, while the volume ratio of the mixture: diatomite granules is 1:5.

Patent RU 2528058 (published 10.09.2014) is known, which describes a biological product based on the strain Bacillus amyloliquefaciens VKPM B-11475, which has a fungicidal effect for protecting grain plants from diseases caused by phytopathogenic fungi. The biological product is obtained by mixing the active ingredient in the form of a culture liquid of the above-mentioned strain with a titer of 2-3⋅10 ⁹ CFU/ml and a carrier in the form of finely dispersed diatomite granules in a volume ratio of 1:3, followed by drying.

In patent RU 2808722 and patent RU 2528058, finely dispersed diatomite, which is a natural sedimentary rock consisting mainly of the remains of diatom algae, is selected as a carrier for immobilizing the active factor of Bacillus amyloliquefaciens VKPM B-13788 and Bacillus amyloliquefaciens B-11475, respectively. Chemically, diatomite consists of more than 80% hydrous silica.

The prior art does not know of the use of the Bacillus subtilis strain VKM B3701D in a composition with antifungal activity for use as a biological fungicide in plant growing.

The objective of the invention is to develop a composition based on Bacillus subtilis VKM B3701D with antifungal activity.

The technical result is a solution to the set problem due to the proposed composition containing Bacillus subtilis VKM B3701D with a titer of 1·10 ⁹ CFU/g and 0.2 M CaSO ₄ ·2H ₂ O, which has a higher antifungal activity compared to a pure culture.

The Bacillus subtilis strain VKM B3701D for the composition with CaSO ₄ 2H ₂ O was isolated from poultry waste by serial dilutions on LB nutrient agar of the following composition: peptone - 10; yeast extract - 5; NaCl - 10; agar - 20. The culture was selected based on the highest deaminating activity, for the detection of which a modified LB nutrient medium was prepared with the addition of 0.1 ml of 0.5% bromothymol blue indicator solution per 1 liter of medium [Senchenkov, VY; Lyakhovchenko, NS; Nikishin, IA; Myagkov, DA; Chepurina, AA; Polivtseva, VN; Abashina, TN; Delegan, YA; Nikulicheva, TB; Nikulin, IS; et al. Whole-Genome Sequencing and Biotechnological Potential Assessment of Two Bacterial Strains Isolated from Poultry Farms in Belgorod, Russia. Microorganisms 2023, 11, 2235. https://doi.org/10.3390/microorganisms11092235].

Cultural and morphological properties of the Bacillus subtilis strain VKM B3701D

Bacillus subtilis VKM B3701D is a rod-shaped, gram-positive, non-motile, spore-forming bacterium. The isolate is a facultative anaerobe.

Physiological and biochemical properties of the strain Bacillus subtilis VKM B3701D

The isolate has nitrate reductase, proteolytic and amylase activities, and is capable of using gelatin, albumin and casein as growth substrates. Bacillus subtilis VKM B3701D produces ammonia when grown on a 3% peptone medium.

The strain utilizes D-glucose, sucrose, maltose, lactose, mannitol, sorbitol, tyrosine, phenylalanine, dehydroxyphenylalanine, isoleucine and lysine.

In turn, the isolate is not able to utilize fructose, histidine, cysteine, threonine, serine, norleucine, glutamine and ornithine. The isolate is not able to use sodium benzoate as a growth substrate.

Bacillus subtilis VKM B3701D is resistant to antibiotics such as amphotericin B - 40 mcg, bacitracin - 0.04 units, optochin - 6 and saponin - 750 mcg, as well as to sodium deoxycholate - 3 mg, the antimycotic ketoconazole - 20 mcg.

Molecular genetic identification of the Bacillus subtilis strain VKM B3701D.

During the molecular genetic identification of the isolate, gene clusters of secondary metabolites characteristic of the Bacillus subtilis species were discovered. These include bacillin, subtilisin, bacilizin, surfactin, bacillibactin, fengycin, sactipeptode, and ratipeptide. Most of the compounds that this strain is theoretically capable of producing are known for their antimicrobial activity.

It was unexpectedly discovered that the Bacillus subtilis strain VKM B3701D has antifungal activity, and when combined with calcium sulfate of the formula CaSO4 2H2O, the antifungal activity increases.

In addition, it is possible to use citro gypsum of the formula CaSO4 2H2O as a component of the claimed composition, which makes it possible to solve the problem of recycling gypsum deposits, which act as a waste product of citric acid production.

Method for obtaining a composition based on Bacillus subtilis VKM B3701D and CaSO ₄ ·2H ₂ O.

The method includes the preparation of nutrient media, daily cultures and sterile water. The prepared daily culture is suspended and added to the nutrient medium. After that, it is cultivated for 48 hours under optimal conditions for the growth of Bacillus subtilis VKM B3701D, namely, 30°C, pH of the medium 7, stirring at 150 rpm.

CaSO ₄ ·2H ₂ O powder is sterilized by autoclaving at 115°C for 30 minutes.

At the end of the cultivation, sterile calcium sulfate powder is added to the Bacillus subtilis VKM B3701D suspension in such a quantity that its concentration is 0.2 M in the resulting mixture, and mixed at 150 rpm for 30 minutes.

The resulting mixture is frozen at minus 40°C for 12 hours, after which it is lyophilized in a vacuum for 17 hours.

As a result, a composition is obtained containing Bacillus subtilis VKM B3701D with a titer of 1 10 ⁹ CFU/g and 0.2 M CaSO ₄ 2H ₂ O.

The invention is characterized by the following figures.

Fig. 1. Scheme of the experimental setup, where: 1) Petri dish; 2) nutrient medium; 3) agar disks with the culture under study; 4) filter paper disk soaked in a suspension of the test culture of the mold fungus.

Fig. 2. Graph of the change in the mean square diameter of a mold fungus in the presence of bacteria, where:

1 - isolated culture of Alternaria brassicicola VKM F-1864 as a control;

2 - culture of Alternaria brassicicola VKM F-1864 in the presence of test culture of Bacillus subtillis VKM B-12;

3 - culture of Alternaria brassicicola VKM F-1864 in the presence of a composition of Bacillus subtilis VKM B3701D with CaSO ₄ ·2H ₂ O;

4 - culture of Alternaria brassicicola VKM F-1864 in the presence of Bacillus subtilis VKM B3701D without CaSO ₄ ·2H ₂ O.

Fig. 3. Graph of the change in the mean square diameter of a mold fungus in the presence of bacteria, where:

1 - isolated culture of Aspergillus unguis BKM F-1754 as a control;

2 - culture of Aspergillus unguis BKM F-1754 in the presence of test culture of Bacillus subtilis VKM B-12;

3 - culture of Aspergillus unguis BKM F-1754 in the presence of a composition of Bacillus subtilis VKM B3701D with CaSO ₄ ·2H ₂ O;

4 - culture of Aspergillus unguis BKM F-1754 in the presence of Bacillus subtilis VKM B3701D without CaSO ₄ 2H ₂ O.

Example 1. Obtaining a composition including Bacillus subtilis VKM B3701D and CaSO ₄ ·2H ₂ O:

1.1. Using the microbiological streak method on a Petri dish, a pure daily culture of the gram-positive bacterium Bacillus subtilis VKM B-3701D is obtained on a dense nutrient medium consisting of 3% microbiological peptone, 2% microbiological agar-agar, and the rest is water.

1.2. Using a microbiological loop, take 1 cm of the microbiological streak of the grown culture of Bacillus subtilis VKM B-3701D obtained according to item 1 and mix with 4 ml of sterile water. After which, the mixture is added to 100 ml of liquid nutrient medium containing 3% microbiological peptone.

1.3. The culture is cultivated for 48 hours at a pH of 7, a temperature of 30°C and with shaking at 150 rpm.

1.4. At the same time, calcium sulfate is prepared by sterilization by autoclaving at a temperature of 121°C for 30 minutes.

1.5. A composition of the microorganism and CaSO ₄ 2H ₂ O is obtained by adding the latter to 100 ml of the culture liquid of Bacillus subtilis VKM B-3701D CaSO ₄ 2H ₂ O in an amount of 27.2 g, which makes up 0.2 mol of the resulting mixture.

1.6. The resulting mixture is stirred at 150 rpm for 30 minutes and

frozen at minus 40°C for 12 hours.

1.8. Lyophilize the mixture for 17 hours in a vacuum.

As a result, a free-flowing powder of a composition is obtained, including Bacillus subtilis VKM B-3701D with a titer of 1 10 ⁹ CFU/g and 0.2 M CaSO ₄ 2H ₂ O in an amount of 1 g.

Example 2. Determination of the antifungal activity of the composition obtained according to Example 1 using the example of the fungal necrotrophic plant pathogen Alternaria brassicicola VKM F-1864, which causes black spot disease in a wide range of hosts, especially in representatives of the genus Brassica, including a number of economically important crops such as cabbage, Chinese cabbage, cauliflower, oilseeds, broccoli and rapeseed. Although it is mainly known as a significant plant pathogen, it also contributes to the development of various respiratory allergic conditions such as asthma and rhinoconjunctivitis.

1) 0.1 g of a composition containing 1 x 10 ⁹ CFU/g of Bacillus subtillis VKM B3701D bacteria and 0.2 M CaSO ₄ x 2H ₂ O, obtained according to Example 1, is added to a liquid nutrient medium consisting of peptone - 10 g; yeast extract - 5 g; NaCl - 10 g and 1000 ml of water. The culture is incubated at 30°C for 48 hours;

2) a reference strain of the bacterium Bacillus subtillis VKM B-12 is prepared in a similar manner, acting as a control culture, and a pure culture of Bacillus subtillis VKM B3701D without calcium sulfate with a titer of 1 10 ⁹ CFU/ml. The cultures are incubated at 30°C for 48 hours;

3) suspensions of cultures obtained according to points 1 and 2 are passaged into sterile Petri dishes with a nutrient medium, the composition of which is calculated as follows: peptone - 10 g; yeast extract - 5 g; NaCl - 10 g; agar - 20 g and 1000 ml of water, using a continuous method - sowing "lawn" - and incubated at 30 ° C for 5 days;

4) after incubation, using a sterile cork drill, cut out discs from the agar with the culture, 10 mm in diameter;

5) The disks are placed 8 at a time in a Petri dish, according to the diagram shown in Figure 1.

6) in the center of the Petri dish, place a paper disk of filter paper with a diameter of 10 mm, moistened with a suspension of spores of the mold fungus Alternaria brassicicola VKM F-1864;

7) The crops are incubated at 25°C. The diameter of the fungal colony is measured every 24 hours for 7 days;

8) for each measurement, the mean square diameter of the mold fungus is calculated using the formula:

(1)

The growth rate of the mean square diameter of the colony is calculated using the formula:

(2)

where K is the colony growth rate, S ₀ is the mean square diameter of the colony at the first measurement, S is the mean square diameter of the colony at the last measurement, t ₀ is the incubation time at the time of the first measurement, t is the incubation time at the time of the last measurement.

The degree of suppression of the growth rate of the mean square diameter of the colony is calculated using the formula:

(3)

where K _k is the growth rate of the culture in the control variant at the end of incubation, and K _o is the growth rate in the experimental variant.

9) to demonstrate the influence of bacterial strains during joint cultivation with the fungus, a graph of the change in the mean square diameter of the mold fungus in the presence of bacteria and a pure culture of the mold fungus is plotted (Figure 2);

10) according to formula 2, the growth rate of the mean square diameter of the mold colony in the control variant is 11.6 mm/day, while in the presence of the test culture Bacillus subtillis VKM B-12 it is 5.3 mm/day. In the presence of a pure culture of Bacillus subtillis VKM B3701D, the growth rate of the mean square diameter of the mold colony is 3.5 mm/day, and for the composition of Bacillus subtillis VKM B3701D with a titer of 1 10 ⁹ CFU/g and 0.2 M CaSO4 2H2O - 2.7 mm/day.

10) Significant antifungal activity is considered to be such a value of the degree of inhibition of the growth of the mold fungus test culture, which exceeds 70%. According to formula 3, the degree of inhibition of the growth of Alternaria brassicicola VKM F-1864 by the test culture Bacillus subtillis VKM B-12 was 54.3%, by the culture Bacillus subtillis VKM B3701D - 70%, and the composition of Bacillus subtillis VKM B3701D with a titer of 1 10 ⁹ CFU/g and CaSO ₄ 2H ₂ O - 76.7%. Consequently, the degree of suppression of the composition of Bacillus subtillis VKM B3701D with CaSO ₄ 2H ₂ O proposed for patenting is higher than that of the test culture obtained from the All-Russian collection of microorganisms Bacillus subtillis VKM B-12 by 22.4%, and by 15.7%, higher than that of the pure culture of Bacillus subtillis VKM B3701D without calcium sulfate. Which proves the increase in antifungal activity with the addition of calcium sulfate.

Example 3. Determination of antifungal activity using the example of the fungal plant pathogen Aspergillus unguis BKM F-1754. Representatives of the genus Aspergillus are widespread in soil and many strains are pathogens of plant diseases. In addition, mold fungi of this genus can cause diseases in humans and animals - aspergillosis:

1) strains of Bacillus subtillis VKM B-12, Bacillus subtillis VKM B3701D and compositions of Bacillus subtillis VKM B3701D with CaSO ₄ ·2H ₂ O are prepared according to points 1, 2, 3 of example 2;

2) the experiment to evaluate the antifungal activity of strains against the mold fungus Aspergillus unguis BKM F-1754 was carried out according to points 4, 5, 6 of example 2;

3) then, using formulas 1-3, the obtained data are processed and the efficiency of the strains is judged. Using formula 1, the mean square diameter of the mold fungus is calculated and growth curves are plotted to demonstrate the effect of bacterial strains when co-cultivated with the fungus (Figure 3);

4) according to formulas 2 and 3, the growth rate of the mean square diameter of the colony of the mold fungus Aspergillus unguis BKM F-1754 in the control variant is 3.42 mm/day, whereas in the presence of the test culture of Bacillus subtillis VKM B-12 - 1.5 mm/day. In the presence of the pure strain of Bacillus subtillis VKM B3701D - 1.0 mm/day. In the presence of a composition containing the strain Bacillus subtillis VKM B3701D with CaSO ₄ 2H ₂ O, the growth rate of the mean square diameter of the colony of the mold fungus is 0.5 mm/day.

Thus, the degree of suppression of the growth of Aspergillus unguis BKM F-1754 by the test culture of Bacillus subtillis VKM B-12 was 56%, by the pure culture of Bacillus subtillis VKM B3701D without CaSO ₄ 2H ₂ O - 70.7%, in the presence of the declared composition of Bacillus subtillis VKM B3701D with CaSO ₄ 2H ₂ O - 85.4%. Consequently, the degree of growth suppression of Aspergillus unguis BKM F-1754 by the composition of the Bacillus subtillis strain VKM B3701D with CaSO ₄ 2H ₂ O proposed for patenting is higher than that of the test culture obtained from the All-Russian collection of microorganisms Bacillus subtillis VKM B-12 by 29.4%, and the pure culture of Bacillus subtillis VKM B3701D by 14.7%.

Thus, the given examples confirm that the task has been solved and the technical result has been achieved.

Claims (1)

A composition with antifungal activity for use as a biological fungicide in plant growing, containing Bacillus subtilis VKM B3701D with a titer of 1⋅10 ⁹ CFU/g and 0.2 M CaSO ₄ ⋅2H ₂ O.