Search for biocontrol agents among endophytic lipopeptide-synthesizing bacteria Bacillus spp. to protect wheat plants against Greenbug aphid (Schizaphis graminum).

Beneficial endophytic bacteria can suppress the development of insect pests through direct antagonism, with the help of metabolites, or indirectly by the induction of systemic resistance through the regulation of hormonal signaling pathways. Lipopeptides are bacterial metabolites that exhibit direct antagonistic activity against many organisms, including insects. Also, lipopeptides are able to trigger induced systemic resistance (ISR) in plants against harmful organisms, but the physiological mechanisms of their action are just beginning to be studied. In this work, we studied ten strains of bacteria isolated from the tissues of wheat and potatoes. Sequencing of the 16S rRNA gene showed that all isolates belong to the genus Bacillus and to two species, B. subtilis and B. velezensis. The genes for lipopeptide synthetase - surfactin synthetase (Bs_srf ), iturin synthetase (Bs_ituA, Bs_ituB) and fengycin synthetase (Bs_fenD) - were identified in all bacterial isolates using PCR. All strains had high aphicidal activity against the Greenbug aphid (Schizaphis graminum Rond.) due to the synthesis of lipopeptides, which was proven using lipopeptide-rich fractions (LRFs) isolated from the strains. Endophytic lipopeptide-synthesizing strains of Bacillus spp. indirectly affected the viability of aphids, the endurance of plants against aphids and triggered ISR in plants, which manifested itself in the regulation of oxidative metabolism and the accumulation of transcripts of the Pr1, Pr2, Pr3, Pr6 and Pr9 genes due to the synthesis of lipopeptides, which was proven using LRF isolated from three strains: B. subtilis 26D, B. subtilis 11VM, and B. thuringiensis B-6066. We have for the first time demonstrated the aphicidal effect of fengycin and the ability of the fengycin-synthesizing strains and isolates, B. subtilis Ttl2, Bacillus sp. Stl7 and B. thuringiensis B-6066, to regulate components of the pro-/antioxidant system of aphid-infested plants. In addition, this work is the first to demonstrate an elicitor role of fengycin in triggering a systemic resistance to S. graminum in wheat plants. We have discovered new promising strains and isolates of endophytes of the genus Bacillus, which may be included in the composition of new biocontrol agents against aphids. One of the criteria for searching for new bacteria active against phloem-feeding insects can be the presence of lipopeptide synthetase genes in the bacterial genome.

The effect of salicylic and jasmonic acids on the activity of SnAGO genes in the fungus Stagonospora nodorum Berk. in in vitro culture and during infection of wheat plants.

RNA interference is a gene silencing mechanism that plays an important role in genetic regulation in a number of eukaryotes. Argonaute (AGO) proteins are central to the complex RNA interference system. However, their role in this mechanism, both in the host plant organism and in the pathogen, has not yet been fully elucidated. In this work, we identified and phylogenetically analyzed the SnAGO1, SnAGO2, SnAGO3, and SnAGO18 genes of the pathogenic fungus Stagonospora nodorum Berk., and analyzed their expression under conditions of infection of plants with varying degrees of resistance to the pathogen. The expression level against the background of plant immunization with the resistance inducers salicylic and jasmonic acids was assessed. In addition, the activity of these genes in the culture of the fungus in vitro was studied under the direct influence of resistance inducers on the mycelium of the fungus. Earlier activation of the SnAGO genes in in vitro culture under the influence of salicylic and jasmonic acids suggests their sensitivity to it. In an in vivo system, plant immunization to induce the accumulation of pathogen SnAGO transcripts was found. At the same time, the SnAGO genes of the fungus S. nodorum, when interacting with plant cells, reacted depending on the degree of host resistance: the highest level of transcripts in the resistant variety was observed. Thus, our data prove that the SnAGO genes of the fungus S. nodorum effectively interact with the host defense system in direct proportion to the degree of resistance of the latter to the pathogen. It was proposed to use the ratio of the transcriptional activity of the fungal reference gene SnTub to the host TaRLI gene as a marker of disease development in the initial period of the infectious process.

[Dynamics of adreneractivity after transfer of myocardial infarction: annual observation].

AIM: To study the change in the -adrenergic reactivity of red blood cell membranes in patients during the first year after acute myocardial infarction. MATERIALS AND METHODS: The study included 25 patients with acute myocardial infarction (AMI) who signed informed consent to participate in the study. The erythrocyte membrane -adrenoreactivity index (-ARM) was determined in venous blood samples upon admission to the intensive care unit, one day after admission, 6 and 12 months after the index MI was transferred using the BETA-ARM-AGAT reagent kit (Agat-Med, Russia). RESULTS: According to the results of dynamics assessment of -APM during the first day, patients included in the study were divided into 2 groups. Group 1 (n=14) included patients who had an increase in -APM in the first day, and group 2 (n=21) included patients in whom -ARM either did not change or decreased. At the time of admission to the hospital in the formed groups, there were no differences in the -APM index and clinical and anamnestic characteristics. A day after hospitalization, the value of -APM in group 1 significantly exceeded the same indicator in group 2 (p=0.02). At the periods of 6 and 12 months, the -APM indices in the groups did not differ. In the 2nd group of patients, the progression of chronic heart failure to one or more functional classes (NYHA) was significantly more often compared with the 1st group. CONCLUSION: The study showed that on the first day in patients with AMI, both an increase and a decrease in the activity of the sympathoadrenal system are possible with a further leveling of these differences over the next year. For a group of patients with decreased activity of sympathoadrenal system on the first day, a more unfavorable course of heart failure in the post-infarction period is characteristic.

[Signal molecules involved in the regulation of the wheat defense response to Septoria nodorum infection].

The response of Triticum aestivum L. to infection by Septoria nodorum Berk, a pathogen causing speckled leaf blotch, was studied. The effect of salicylic acid (SA) and jasmonic acid (JA) signal molecules, as well as chitooligosaccharides (COSs) with different acetylation degrees (ADs), on the accumulation of hydrogen peroxide (Н2О2) in wheat leaves and the pathogenesis-related (PR) proteins of oxalate oxidase (AJ556991.1), peroxidase (TC 151917), and proteinase inhibitor (EU293132.1) was investigated. Treatment with the signal molecules inhibited S. nodorum growth and stimulated Н2О2 accumulation, as well as PR gene expression. SA and COS with 65% AD are found to be more efficient in Н2О2 induction and elevation of the transcriptional level of the oxalate oxidase and peroxidase genes. At the same time, JA and COS with 30% AD stimulated transcription of the proteinase inhibitor gene. The results suggest the existence of differential means of defense response induction by signal molecules with more prospects for the regulation of plant immunity.

[Inhibition of IAA oxidase activity of wheat anionic peroxidase by chitooligosaccharides].

The study demonstrated that chitooligosaccharides with a molecular weight of 5­10 kDa and a degree of acetylation of 65% exhibited an auxin-like effect in wheat plants and also played an important role in regulating the activity of polysaccharide (chitin)­specific anion isoenzymes of peroxidase oxidizing indole acetic acid. Changes in the kinetic parameters of the interaction of the wheat anionic chitin-specific peroxidase with pI ∼3.5 with chitin oligomers in the presence of indoleacetic acid were pH-depended and indicated that chitooligosaccharides significantly impair the ability of the enzyme for oxidation at pH levels of 4.2 and 6.0. It can be assumed that chitooligosaccharides not only induce protective plant systems but also increase the accumulation of auxin in plant tissues, thus adversely affecting a number of components of the plant protective system against pathogens.

[Regulation of peroxidase activity under the influence of signaling molecules and Bacillus subtilis 26D in potato plants infected with Phytophthora infestans].

The influence of sequential exposure of 5 x 10(-5) M salicylic acid (SA) or 1 x 10(-7) M jasmonic acid (JA) and endophytic bacterium Bacillus subtilis strain 26D on peroxidase activity, transcription of the M21334 isoperoxidase gene from potato (Solarium tuberosum L.), and the formation of resistance to the infective agent of potato blight Phytophthora infestans (Mont.) de Bary was studied. It was found that individual application of JA or Bacillus subtilis 26D and sequential application of SA and B. subtilis 26D were the most effective in protecting plants against pathogens, while sequential application of JA and B. subtilis 26D drastically suppressed plant resistance. The results suggest the need for strict compliance with regulations when using SA and JA, as well as biological products based on living bacteria as modern plant protection products with immunomodulatory properties that trigger specific signaling pathways, which often interfere with each other.

[Effect of chitooligosaccharides with different degrees of acetylation on the activity of wheat pathogen-inducible anionic peroxidase].

The effect of chitooligosaccharides (CHOSs) with a molecular weight of 5-10 kDa and a degree of acetylation (DA) of 65 and 13% at a concentration of 1.0 mg/L on the expression of the TC151917 gene, which encodes wheat anionic peroxidase, and the activity of "anionic" isoperoxidases in common wheat plants infected with Septoria nodorum Berk.--the causative agent of septoriosis. Treatment with CHOSs with a 65% DA and infection promoted the transcription of the anionic peroxidase gene and increased the enzymatic activity of the anionic peroxidase with an isoelectric point of 3.5 in soluble and ion-bound to cell walls protein fractions. Chitooligosaccharides with a 13% DA change these parameters to a lesser extent. These data suggest the importance of the degree of acetylation of CHOSs in the development of immune responses of wheat with the involvement of peroxidases.

[The effect of 1-methylcyclopropene on the components of pro- and antioxidant systems of wheat and the development of protective reactions in fungal pathogenesis].

The effect of 1-methylcyclopropene (1-MCP), which inhibits the reception of ethylene, on the following has been studied: hydrogen peroxide generation, oxalate oxidase activity, peroxidase activity, catalase activity, and lignin accumulation in infected leaves of soft spring wheat (Triticum aestivum L.) breeds that differ in their resistance to the causative agent of leaf blotch Septoria nodorum Berk. A decrease in the development of leaf blotch in wheat leaves under the influence of 1-MCP was, on one hand, followed by an inhibition of catalase activity; on the other hand, it was accompanied by an increase in oxalate oxidase and peroxidase activity, as well as an accumulation of H2O2 in tissues and lignin in the infected zone. The role of the ethylene reception system in the plants' protective response to infection with a hemibiotrophic pathogen, the causative agent of leaf blotch, is discussed.

[RNA-silencing of anionic peroxidase gene decreases the potato plant resistance to Phytophthora infestans (Mont.) de Bary].

Transformed potato (Solanum tuberosum L.) plants expressing the antisense-fragment of M21334 gene were estimated. In transgenic plants the decrease of anionic isoperoxidase pI - 3.5 activity was detected. So, the data testify that M21334 gene encodes this isoperoxidase. Decrease of lignin accumulation and dramatic decline of resistance of transgenic potato plants to the late blight agent Phytophthora infestans emphasize an importance of isoperoxidase pI - 3.5 in defense reaction against late blight.

[Relationship between the aggressiveness and catalase activity of Septoria nodorum berk. in wheat].

A comparative study of hydrogen peroxide (H202) generation, the character of a fungal catalase gene expression, and the catalase activity in wheat plants, infected with Septoria nodorum Berk. strains differing in their aggressiveness, has been carried out. The decreased intensity of H202 accumulation in infected tissues, influenced by an aggressive S. nodorum strain and caused by the enhanced transcriptional activity of the fungal catalase gene and the heightened synthesis of its product, has been revealed to be more expressed compared to a similar decrease influenced by a less aggressive strain. An assumption was made that the expression activity of the catalase gene and, therefore, the activity ofcatalase involved in the regulation of the H202 content in the infected zone represent important factors providing high.aggressiveness and pathogenicity of S. nodorum.

[Comparative efficacy of pharmacoinvasive strategy of myocardial reperfusion and primary angioplasty in patients with acute ST elevation myocardial infarction].

In order to assess comparative efficacy of pharmacoinvasive strategy of myocardial reperfusion and primary angioplasty in ST elevation acute myocardial infarction 289 patients were randomized at prehospital stage within first 6 hours of the disease into 2 groups: primary angioplasty (group 1), and prehospital thrombolysis with subsequent rescue or delayed angioplasty depending on efficacy of thrombolysis. We analyzed clinical and anamnestic characteristics of patients, efficacy of reperfusion measures, dimensions of myocardial necrosis, and clinical course of the disease. Pharmacoinvasive myocardial reperfusion with prehospital thrombolysis compared with primary coronary angioplasty decreased time of myocardial ischemia (224.65 +/- 71 vs. 278 +/- 184 min, p < 0.03), increased rate of achievement of TIMI grade 3 flow after percutaneous coronary intervention (80.5% vs. 71.4%, p = 0.002) and more effectively preserved left ventricular ejection fraction (60.0 +/- 14.9% vs. 54.9 +/- 12.3%, p < 0.01). Prehospital thrombolysis before coronary angioplasty compared with primary angioplasty was associated with lower rate of development of no-reflow syndrome (1.4% vs. 11.6%, p < 0.003).

[Intrathecal immunoglobulin production in the diagnosis and differential diagnosis of multiple sclerosis].

The evaluation of diagnostic significance of different immunological tests for intrathecal immunoglobulin production is summarized on the historical basis of investigation of patients with inflammatory, demyelinating and other neurological disorders. The assessment of cerebrospinal fluid lost its previous significance in the 2010 revision of diagnostic criteria for multiple sclerosis. Nowadays, it is used only for the diagnosis of primary progressive multiple sclerosis. Nevertheless, the requirements of the analysis of the cerebrospinal fluid are increasing due to subtle, subclinical and atypical cases of multiple sclerosis as well as undetermined demyelinating disorders. Intrathecal humoral immune response may be pathogenic in multiple sclerosis as suggest immunological data and effectiveness of anti-B cells treatment. Based on these tests, it is useful, to differentiate subgroups of patients and to evaluate different effects of treatment in perspective.

[About development of operativeness of medical information data system of central military clinical hospital n. a. N.N.Burdenko].

The authors presented the data about the development of operativeness of medical automated information data system of CMCH n. a. N.N.Burdenko with developing and formation of control center of hospitalization, data processing center, situation room and central archives of diagnostic data. It significantly improved diagnostic and treatment processes in hospital medical and preventive association. Consolidation of dependent prevention and treatment facilities of Moscow Region will provide effective administration of medical supply of attached cohort, administration of hospital association of CMCH n. a. N.N.Burdenko and medical service of the Armed Forces of Russian Federation, in general.

Acetylation degree of chitin in the protective response of wheat plants.

Influences on the acetylation degree of chitin manifested by proteins from cultural filtrates of strains of the fungus Septoria nodorum different in aggressiveness and of extracts from leaves of the susceptible (Triticum aestivum) and resistant (Triticum timopheevii) wheat plants infected with these strains were studied. Chitin deacetylase was found among the extracellular proteins of the fungus. Its activity was higher in the aggressive strain of the fungus than in the non-aggressive one, and this suggested that this enzyme could play an important role in the further formation of compatible relationship of the pathogens with the plants. Protein extracts from the susceptible wheat seedlings infected with the septoriosis agent also contained a component decreasing the acetylation degree of chitin. Protein extracts from the resistant wheat seedlings increased the chitin acetylation degree. It is supposed that this can be a pattern of the plant counteracting the action of chitin deacetylases of the pathogen.

[Plant growth promoting microorganisms as alternative to chemical protection from pathogens (review)].

The review analyses data on physiological and biochemical influence of rhizospheric and endophytic microorganisms promoting plant growth (PGPR-plant growth promoting rhizobacteria) on induced resistance of plants and the possibility of its use in plant cultivation to protect crops from pathogens and phytophages. Resistance of plants provided by PGPR due to their endosymbiotic interrelationships is directly achieved because they produce peptide antibiotics and hydrolases ofchitin and glucan and also because plants form their own system of induced resistance, followed by changes in the balance of defensive proteins, phytohormones, and pro-/antioxidant status.

Structural-functional features of plant isoperoxidases.

Current data on structural--functional features of plant peroxidases and their involvement in functioning of the pro-/antioxidant system responding to stress factors, especially those of biotic origin, are analyzed. The collection of specific features of individual isoforms allows a plant to withstand an aggressive influence of the environment. Expression of some genes encoding different isoperoxidases is regulated by pathogens (and their metabolites), elicitors, and hormone-like compounds; specific features of this regulation are considered in detail. It is suggested that isoperoxidases interacting with polysaccharides are responsible for a directed deposition of lignin on the cell walls, and this lignin in turn is concurrently an efficient strengthening material and protects the plants against pathogens.

[Salicylic and jasmonic acids in regulation of the proantioxidant state in wheat leaves infected by Septoria nodorum Berk].

Influence of mediators of the signal systems of salicylic (SA) and jasmonic (JA) acids and their mixture on reactive oxygen species' (ROS) (superoxide radical O2*- and H2O2) generation and activity of oxidoreductases (oxalate oxidase, peroxidase and catalase) in leaves of wheat Triticum aestivum L. infected by Septoria leaf blotch pathogen Septoria nodorum Berk. has been studied. Presowing treatment of seeds by SA and JA decreased the development rate of fungus on wheat leaves. SA provided earlier inductive effect on production of O2*- and H2O2 compared with JA. The protective effect of the salicylic and jasmonic acids against Septoria leaf blotch pathogen was caused by activation of oxalate oxidase, induction of anion and cation peroxidases, and decrease of catalase activity. Ability of compounds to stimulate ROS in the plant tissues can be used as criteria for evaluation of immune-modulating activity of new substances for protection of the plants.

[Influence of Ca2+ ions on metabolism of active oxygen species in combined cultures of wheat calluses with the fungus Tilletia caries].

The effect of Ca2+ on morphophysiological parameters of calluses of wheat Triticum aestovum L., the level of active oxygen species, and the activity of oxalate oxidase, peroxidase, and catalase is investigated in the case of infestation with the fungus Triticum aestivum causing ball smut. The concentration of O2-, H2O2, and activity of oxidoreductases (oxalate oxidase, peroxidase, and catalase) depends on the content of Ca2+ ions in the culture medium of calluses. The increase in the concentration of Ca2+ ions in the culture medium led to higher structuring of calluses, induction of activity of oxalate oxidase and of some forms of peroxidase, and to accumulation of active oxygen species. These changes contributed to inhibition of development of the fungus. Discovery of such dependence agrees with the role of calcium as the intermediary in biochemical reactions related to the formation of the protective response of plant cells in case of infestation.

[Molecular characteristics of the chitin-binding peroxidases of plants].

The chitin-binding ability of isoperoxidases isolated from 23 plants of different species was studied. The activation of peroxidases in a protein extract in the presence of this polysaccharide was found for 14 of the studied plants. Anionic isoperoxidases were shown to be sorbed on chitin and eluted from them with 1 M NaCl for 16 of the plant species. Cationic isoforms of the peroxidases of some species of the Fabaceae and Cucurbitaceae plant families also bound to chitin. An immunochemical similarity was found between the chitin-binding isoperoxidases of taxonomically distant plant species (the Pomaceous, Fabaceae, and gourd families). Moreover, a high homology of the molecular structures of the polysaccharide-binding sites was revealed for the anionic peroxidases of rice, wheat, oat, zucchini, cucumber, and radish. We propose the existence of a special class of plant peroxidases that bind with polysaccharides (chitin) and participate in the protective reactions of plants against pathogens.

[Hydrogen peroxide production in wheat leaves infected with the fungus Septoria nodorum Berk. strains with different virulence].

The effect of two strains of the phytopathogenic fungus Septoria nodorum Berk. of different virulence on the intensity of local generation of hydrogen peroxide in common wheat leaves and the role of oxidoreductases in this process was studied. Differences in the pattern of hydrogen peroxide production in wheat plants infected with high- and low-virulence pathogen strains have been found. The low-virulent S. nodorum strain caused a long-term hydrogen peroxide (H2O2) generation in the infection zone, whereas the inoculation of leaves with the highly virulent strain resulted in a transient short-term increase in the H2O2 concentration at the initial moment of contact between the plant and the fungus. It was shown that the low level of H2O2 prOduction by plant cells at the initial stages of pathogenesis facilitates S. nodorum growth and development. The decrease in the H2O2 concentration induced by the highly virulent S. nodorum strain is determined by inhibition of the oxalate oxidase activity in plant tissues and by the ability of the fungus to actively synthesize an extracellular catalase. The pattern of hydrogen peroxide generation at the initial stages of septoriosis may serve as an index of virulence of S. nodorum population.