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Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2019, Vol. 54, № 1, p. 178-185.
(how to cite this article)

doi: 10.15389/agrobiology.2019.1.178eng

UDC 579.64: 632.9

Acknowledgements:
This work was performed according to the state assignment No. 075-00376-19-00 of the Ministry of Science and Higher Education of the Russian Federation within the framework of the research on subject No. 0686-2019-0013.

 

ISOLATION AND CHARACTERIZATION OF ANTIFUNGAL
METABOLITES OF Bacillus subtilis STRAINS BZR 336G AND BZR 517 USING THE MODIFIED BIOAUTHOGRAPHY METHOD

Т.М. Sidorova, А.М. Asaturova, A.I. Homyak, N.S. Tomashevich

All-Russian Research Institute of Biological Plant Protection, PO box 39, Krasnodar, 350039 Russia, e-mail 0166505@mail.ru (✉ corresponding author), biocontrol-vniibzr@yandex.ru, HomyakAI87@mail.ru, tom-s2@yandex.ru

ORCID:
Sidorova Т.М. orcid.org/0000-0003-4281-5278
Homyak A.I. orcid.org/0000-0001-9360-2323
Asaturova А.М. orcid.org/0000-0002-0060-1995
Tomashevich N.S. orcid.org/0000-0002-7297-5929

Received October 17, 2017

 

The ability of B. subtilis to produce a large number of biologically active metabolites of diverse structure and properties can largely determine its fungicidal effect against particularly dangerous pathogenic fungi. In this regard, we have studied the fungicidal activity and properties of the antifungal metabolites of active strains B. subtilis BZR 336g and BZR 517. The approach we used, inсluding purification by extraction with ethyl acetate, thin-layer chromatography (TLC), UV fluorescence, tests with detection reagents, spectroscopy and bioautography, showed the potential of the studied B. subtilis strains to accumulate the complex of active metabolites exhibiting antifungal effect from fungistatic to fungicidal. Bioautographic method was modified by use of the causative agents of harmful diseases, F. oxysporum var. orthoceras and Alternaria sp. as test organisms, which allowed us to assess in vitro the antifungal activity. Patterns of the antifungal compounds differed between the strains in TLC mobility and UV fluorescence. The impact on fungal growth also differed, from slight changes in mycelium growth to total suppression. In strain B. subtilis BZR 336g the most pronounced inhibition of F. oksysporum var. orthoceras BZRP1 growth was in bands with Rf 0.39 and 0.96. Significant Alternaria sp. BZRP8 growth inhibition was also observed in two bands, with Rf 0.42 and 0.96. B. subtilis BZR 517 synthesizes metabolites exhibiting fungitoxic properties in four bands with Rf 0.42, 0.84. 0.92 and 0.96 against F. oxysporum var. orthoceras BZRP1, and in two bands with Rf 0.42 and 0.96 against Alternaria sp. BZRP8. It is known that, in addition to the direct action, surfactins and fengicins of B. subtilis prevent the adhesion of competitive microbes on the plant. In out tests, spraying TLC plates with distilled water revealed bands with hydrophobic properties (non-wetting white zones), which can enhance the antagonistic effect of B. subtilis. Test with detection reagents visualized the presence of aromatic amines, phenols, phenolic steroids, methyl hydrazines, that is, compounds that probably play a certain role in the bioactivity manifestation. The weak staining of the metabolites with ninhydrin, as well as the absence of characteristic precipitation during acidification of the culture medium or adding ammonium sulfate, may indicate the presence of only minor amounts of amino acids and peptides. Spectrophotometrically we detected absorption peaks at 205-217 nm wavelengths, which indirectly evidences the presence of polyene structures. Our data allow us to hypothesize mechanisms underlying antifungal effects in the studied B. subtilis strains. These effects may be due to hydrolysis of phenolic compounds, since the detecting reagents indicate various phenolic derivatives, and, according to many researchers’ opinion, active strains, when interacting with a phytopathogen, produce hydrolytic enzymes. As a result, the active groups of phenols can be released, which enhances the antifungal activity of the synthesized metabolites via effect on both fungal cell walls and other cell structures.

Keywords: Bacillus subtilis, microbiological preparations, antifungal metabolites, fungitoxicity, thin-layer chromatography, bioautography, detection reagents, phenolic compounds, polyene structures.

 

 

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