Prikladnaâ biohimiâ i mikrobiologiâ

Phytopathogenic fungi pose a threat to food security, limiting the biological potential of agricultural crops and reducing the quality of products. New plant protection methods based on natural systemic and cellular phytoimmunity are being developed to date, where a unique mechanism, described by the term “RNA interference” (RNAi), occupies a special place. RNAi regulates the expression of target genes in a homologically dependent manner and, with the involvement of a protein complex designated as RISC (RNA-induced silencing complex), on the one hand, it protects plants from pathogens, but on the other hand, pathogens use it as a virulence factor. Cases of bilateral exchange of small RNAs between plants and pathogens affecting them through extracellular vesicles have been described. This review discusses the role of small RNAs, as well as DCL, AGO, and RdR proteins, in the infection of plants with pathogenic fungi and oomycetes, and the prospects for using RNAi in the development of environmentally friendly, modern plant protection products.

The specific features of the reactivation of “non-culturable” (NC) Mycobacterium tuberculosis (MTB) cells for the following propagation in liquid medium were defined, as well as the approach for the quantification of these cells by culture methods was suggested. When NC cells obtained in vitro were inoculated into standard Sauton’s liquid medium, a prolonged (up to 20 days) lag-phase is observed, in which no cell propagation is occurred. During the lag-phase, dormant cells secrete substances of unknown nature that inhibit or completely arrest the reactivation and growth of cultures when initial dormant cells concentration is above 10⁷–10⁸ cells/mL. Addition of meat-peptone broth (3.25 g/L) into a standard Sauton’s medium with a 10-fold reduced concentration of glycerol significantly stimulated the reactivation and propagation of the initially dormant cells inoculated at a concentration above the threshold. This modification of the medium composition made it possible to detect up to 10³ times more cells in the MTB population by the method of the Most Probable Number (MPN) of cells. Increased number of the detected dormant cells in the population (up to an average of 2.5 orders of magnitude) was also achieved by adding to the reactivation medium recombinant protein RpfB (5 ng/mL), a protein of the Rpf family – resuscitation promoting factor of dormant bacteria. Perhaps the action of a Rpf enzyme is related to the products of its enzymatic activity, since an increased MPN value in the dormant cells population was also observed when the products of mycobacteria peptidoglycan hydrolysis obtained by the coaction of RpfB and endopeptidase RipA were added. The addition of sonicated peptidoglycan fragments at a concentration up to 1 μg/mL had a similar effect. The obtained results may be used as approaches to optimize liquid media composition and culture conditions aiming to identify in clinical samples the pathogen of tuberculosis remain in “non-culturable” state.

Plant roots secrete various organic substances into the rhizosphere, which are a source of nutrition for microorganisms and largely determine the nature of plant-microbe interactions. The composition of the main fractions of root exudates in ten modern varieties of wheat was determined: amino acids, organic acids and sugars. Reliable qualitative and quantitative differences between varieties for individual components of exudates were revealed, which determined the peculiarities of cultivar clustering on this trait. Relationships between exudation and the effectiveness of plant interaction with the growth-promoting rhizobacterium Pseudomonas fluorescens SPB2137 and the phytopathogenic fungus Fusarium culmorum 30 in laboratory systems, as well as with the resistance of varieties to diseases in the field, were found. The number of P. fluorescens SPB2137 in the root zone positively correlated with the amount of many amino acids, as well as maltose, secreted by the roots. The stimulating effect of rhizobacteria on root growth positively correlated with the amount of released glucose and melibiose. The relationship between the nature of root exudation and root colonization or the susceptibility of varieties to F. culmorum 30 was not found. The analysis of correlations between the incidence of wheat varieties in the field and the intensity of exudation of certain substances, as well as with the biocomposition index of amino acid exudation, was carried out. The role of root exudate components in the formation of effective plant-microbial systems is discussed.

A new method for extracting DNA from plants is proposed, using the example of wild grapes Vitis amurensis Rupr., for further preparation of libraries for metagenomic analysis. The method is based on the isolation of DNA by an inexpensive CTAB method with an additional stage of DNA purification using silica spin columns (CTAB spin method). A comparative analysis of the results of metagenomic analysis of endophytes on DNA isolated using the proposed CTAB-spin method and using the commercial set ZymoBIOMICS DNA Miniprep (Zymo Research). It was found that when using the CTAB-spin method, the number of sequences of the 16S rRNA site and the diversity of bacterial genera were 2.8 and 1.2 times greater, respectively, than when using the ZymoBIOMICS kit. At the same time, the number of sequences of the internal transcribed spacer 1 (ITS1) and the biodiversity of endophytic fungi did not differ significantly during DNA extraction by two methods. Thus, the proposed method of DNA isolation for metagenomic analysis is an available and effective alternative to commercial kits for the isolation of plant DNA for new generation sequencing methods.

A study was made of the response of a sensor element based on polydiphenylenephthalide to a change in the composition of the air medium during the cultivation of Enterobacter asburiae UOM 3 bacteria in a liquid nutrient medium. Registration of changes in the resistance of the sensor element in response to volatile organic compounds released by bacterial cells was carried out by measuring the current-voltage characteristics at specified time intervals. The results showed the relationship between the number of bacteria and the change in the resistance of the sensor element, due to the release of waste products of microorganisms. With an increase in the titer of bacteria by 3 orders of magnitude, the resistance of the sensor under the influence of volatile organic compounds accumulated in the medium decreased by 2 orders of magnitude. It is assumed that sensors based on polydiphenylenephthalide can be used to determine the presence of bacteria in various materials and media.

Phthalic acid esters are integral components of modern plastic products and packaging materials, which causes significant contamination of food products and the environment, leading to the need for simple productive monitoring methods. The article presents a rapid enzyme-linked immunosorbent assay (ELISA) for the determination of dibutyl phthalate (DBP) in fruit juices, based on the competitive interaction between free and bound antigen for the binding sites of specific antibodies. The analytical characteristics of the method were studied in various kinetic regimes of the competition stage. Optimal conditions have been established to ensure the minimum detection limit and high measurement accuracy. The duration of the competitive stage of ELISA was chosen 30 min; the range of determined concentrations of DBP was from 0.37 to 68.34 ng/mL with a detection limit of 0.08 ng/mL. The efficiency of the proposed ELISA for testing fruit juices was shown for the chosen DBP extraction mode.