RU2802234C1 - Bacterial strain bacillus subtilis subspecies subtilis krd-20 is a producer of fungistatic lipopeptides - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: Bacillus subtilis bacterial strain subtilis krd-20 subspecies, a producer of fungistatic lipopeptides, can be used to obtain a fungicidal biological product for protecting agricultural plants from phytopathogenic fungi and for the biosynthesis of lipopeptides, biosurfactants surfactin and fengycin.

EFFECT: strain was deposited in the All-Russian Collection of Microorganisms under the number VKM B-3516D.

1 cl, 4 dwg, 3 ex

Description

SUBSTANCE: invention relates to biotechnology and can be used in plant protection against phytopathogenic microscopic fungi, and also as a producer of surfactin and fengycin lipopeptides.

At present, diseases of plants with phytopathogenic microscopic fungi are becoming an increasingly acute problem, the most harmful are Fusarium diseases of above-ground organs and plant roots. Mushrooms secrete mycotokins, which adversely affect not only the plants themselves, but also human and animal health when consumed. At the moment, the most common method of combating phytopathogens is the use of chemical fungicides, however, their significant drawback is the accumulation of components in environmental objects and accumulation in agricultural products produced on the basis of these plants. Therefore, recently, biofungicides developed on the basis of microorganisms that have antagonistic activity against pathogenic fungi and inhibit their growth have attracted more and more attention.

Known strains of bacteria Bacillus pumilus BKM CR-333D (RF Patent No. 1817875, publ. 05/20/1995) and Bacillus atrophaeus VKPM-11474 (RF Patent No. 2570624, publ. 10.12.2015). The disadvantage of these strains is the lack of biological activity, which manifests itself in antagonism to phytopathogenic fungi.

Known bacterial strain Bacillus subtilis M-22 (RF Patent No. 2084152, publ. 20.07.1997), is a producer of the biopesticide Gamair, which inhibits the growth and development of fungal and bacterial microflora on plant debris and the surface of root crops. The disadvantage of this strain is the impact on pathogens only vegetable crops. Bacillus subtilis subspecies subtilis krd-20 strain showing; antagonistic activity on such phytopathogenic fungi as Fusarium oxysporum, Trichoderma harzianum using lipopeptide biosynthesis.

Closest to the proposed is a bacterial strain producer of surfactin Bacillus subtilis 0017 (RF Patent No. 2270858, publ. 27.02.2006). However, it can only be used to obtain a biogenic surfactant - surfactin.

The Bacillus subtilis subspecies subtilis krd-20 strain produces fungistatic lipopeptides such as surfactin and fengycin.

The technical result of the present invention is the isolation of a new bacterial strain that has antagonistic activity against phytopathogenic fungi and produces lipopeptides (biosurfactants) surfactin and fengycin, and also does not adversely affect the environment and reduces the development of resistance of pathogenic organisms to fungicides.

The technical result is achieved by the proposed strain of Bacillus subtilis subspecies subtilis krd-20 isolated from the soil of leached wheat agrocenosis chernozem in the conditions of the Krasnodar region. The strain was deposited in the All-Russian Collection of Microorganisms of the IBPM. G.K. Scriabin RAS under the registration number VKM B-3516D.

The essence of the invention is illustrated by the drawing, where figure 1 shows a photograph - reflecting the inhibition of aerial and substrate mycelium by strains of bacteria of the genus Bacillus; Figure 2 is a photograph showing the antifungal activity of VKM antagonist strain B-3516D against Trichoderma harzianum on potato glucose agar; The figure 3 shows a photograph showing the antifungal activity of the VKM B-3516D antagonist strain against Pleurotus ostreatus on potato-glucose agar; The figure 4 shows a graph of the peaks of molecules reflecting the spectra of lipopeptides in acetonitrile after extraction from the precipitate of the culture fluid of Bacillus subtilis subspecies subtilis krd-20 on MALDI-TOF-MS, where A - 1022, 1036, 1044, 1058, 1060, 1074 - surfactin class, and B - 1464, 1478, 1492, 1506 - fengycin class.

The claimed strain has the following characteristics.

Cultural and morphological features

Cells of the strain Bacillus subtilis subspecies subtilis krd-20, the attachment contains a certificate of deposit of the bacterial strain, are rods 0.8-0.9 μm × 1.7-2 μm in size, gram-positive, have peritrichous flagella, movable. Spores are oval, not exceeding the size of the cell, located centrally.

The bacterial strain was grown on MPA and potato dextrose agar at 28°C for 2-5 days. On MPA, colonies 6-7 mm in diameter are dense, irregularly shaped, whitish, matte, the edge of the colony is wavy, not even. On potato-dextrose agar, colonies 5-10 mm in diameter are dense, irregularly shaped, dull, milky-white in color, the edge of the colony is wavy, not even.

Physiological features: Bacillus subtilis subspecies subtilis krd-20 strain is an aerobic bacterium, has catalase activity, breaks down starch, glycogen. The minimum growth temperature is 10°C, the maximum growth temperature is 47°C, the optimal range is 28-32°C, at pH values from 4.5 to 8.5 with an optimum of 7.0-7.5.

The composition of the culture medium (g/l): corn extract - 5.0, beet molasses - 10, KH ₂ PO ₄ - 0.25, MgSO4 * 7H ₂ O - 0.5, pH 6.8.

A pure culture of the strain Bacillus subtilis subspecies subtilis krd-20 is stored in test tubes on a slant agar medium (potato-glucose agar or dry nutrient agar for cultivating microorganisms). Storage temperature +3-+8°С. Shelf life 12 months.

In more detail the essence of the described invention is disclosed in the examples below.

Example 1. Manifestation of antagonistic activity by the counter-growth method.

The evaluation of fungistatic properties was carried out in vitro on potato dextrose agar (PDA medium) in Petri dishes by the counter-growth method. Test cultures were inoculated at a distance of 30 mm opposite each other and incubated at 28°C for five days. After the incubation period, the degree of antagonism was analyzed by the size of the growth inhibition zone (mm).

In the course of the study, in order to select potential antagonists that inhibit Fusarium oxysporum, 50 endospore-forming isolates and morphologically corresponding to representatives of the genus Bacillus were isolated from the soil. Among these isolates, 6 strains showed a significant fungistatic effect. The size of the growth retardation zone varied from 5 to 7 mm +/- 1. Two forms of the fungistatic effect were observed. Strains 5-6 formed a zone of growth inhibition with inhibition of both aerial and substrate mycelium (Fig. 1). Strain 5 is assigned to Bacillus Amyloliquefaciens and strain 6 to Bacillus subtilis subspecies subtilis. Further, the strain was identified as Bacillus subtilis subspecies subtilis krd-20 and deposited under the number VKM B-3516D

Example 2 Another method for determining the fungistatic effect of peptide metabolites was carried out by the agar well diffusion method. To do this, Trichoderma harzianum was inoculated into the center of the dish on the surface of potato-glucose agar, holes were punched at a distance of 20 mm (diameter 5 mm), 50 μl of the sample, previously filtered through a sterile filter with a pore diameter of 0.22 μm, was introduced. Incubation was carried out at 25°C for five days.

To select potential antagonists that suppress Trichoderma harzianum, about 70 bacterial strains belonging to the genus Bacillus species were isolated from the soil. Among these isolates, strain VKM B-3516D showed the greatest antagonistic effect against Trichoderma harzianum (Fig. 2) and did not suppress the growth of Pleurotus Ostreatus (Fig. 3).

Example 3 Mass Spectrometry Analysis (MALDI-TOF-MS)

The acetonitrile solution after extraction of the precipitate was subjected to MALDI-TOF mass spectrometric analysis using an ULTRAFlex2 mass spectrometer. The culture medium extract without inoculation was used as a control. Mass spectra were obtained on a mass spectrometer with a 337 nm nitrogen laser at a pulse frequency of up to 20 Hz. A 0.5 μl sample was mixed on the target with an aliquot of a solution of 20% acetonitrile containing 0.1% trifluoroacetic acid and 20 mg/ml 2,5-dihydroxybenzoic acid and air dried. Mass spectrometers were recorded in the range of 600-2000 m/z. To identify molecules, the peak masses were compared with those previously published in the literature (Sarangi, 2009; Pyoung, 2010).

Analysis of fungicidal lipopeptides. Lipopeptides precipitated from the culture liquid after extraction with acetonitrile were studied by time-of-flight mass spectrometry. MALDI-TOF mass spectrometric analysis of a solution of lipopeptides in acetonitrile revealed two clusters of molecules detected at m/z 1022, 1036, 1044, 1058, 1060, 1074 (surfactin class) and 1464, 1478, 1492, 1506 (fengycin class) (Fig. . 4). These peaks differed by 14 Da, indicating the presence of homologous molecules having different fatty acid chain lengths (CH ₂ =14 Da). At the same time, no peaks corresponding to iturin were detected.

The MALDI-TOF spectrum reveals the predominance of the fengycin profile over the surfactin profile by about two times. Previously, other authors presented the spectra of these lipopeptides, the profiles of which had inverse ratios, i.e. surfactin prevailed over fengycin (Sujata, 2016.).

The conclusion about the peptide nature of these substances was made on the basis that they were isolated by precipitation with ammonium sulfate followed by a qualitative reaction for α-amino acids with ninhydrin. The applicability of the ninhydrin reaction has been established by a number of researchers (Khan, 1989; Friedman, 2004).

To isolate lipopeptides from the culture liquid, we used the precipitation method in a saturated ammonium sulfate solution (Alajlani, 2016).

When determining the antifungal activity by the method of diffusion in agar of the peptide fraction obtained by the method of acid precipitation, sterile distilled water was used as a control, in the case of a salt precipitate, a solution of ammonium sulfate 1.6 M.

Sources taken into account:

1. Pyoung K., Ryu J., Kim Y, Ch Y. Production of biosuractant lipopeptides iturin A, fengycin and surfactin a from bacillus subtilis CMB32 for control of Colletotrichum gloeosporioides. J. Microbiol. Biotechnol. 2010. 20(1), 138-145.

2. Sarangi N.P. Athukorala, W.G. Dilantha Fernando, and Khalid. Y. Rashid. Identification of antifungal antibiotics of Bacillus species isolated from different microhabitats using polymerase chain reaction and MALDI-TOF mass spectrometry. Can. J. Microbiol. 55:1021-1032 (2009). doi:10.1139/W09-067.

3. Sujata S., Sanket J., Geetha S.J. Lipopeptide production by Bacillus subtilis R1 and its possible applications. Brazilian journal of microbiology. 2016. V. 47, P. 955-964.

4. Khan A. A. Studies of the kinetics and mechanism of interaction of α-aminoacids with ninhydrin // J. Indian Chem. soc. - 1989. - Vol. 66, no. 7. - P. 454-456.

5. Friedman A. Applications of the ninhydrin reaction for analysis of amino acids, peptides, and proteins to agricultural and biomedical sciences // J. Agric Food Chem. - 2004. Feb. 11;52(3):385-406. doi:10.1021/jf030490p.

6. Alajlani M., Shiekh A., Hasnain S., Brantner A. Purification of Bioactive Lipopeptides Produced by Bacillus subtilis Strain BIA // Chromatographia (2016) 79:1527-1532 DOI 10.1007/s10337-016-3164-3.

Claims (1)

The bacterial strain Bacillus subtilis subspecies subtilis krd-20 VKM B-3516D is a producer of fungistatic lipopeptides, for the biosynthesis of lipopeptides - biosurfactants surfactin and fengycin.