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.

[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.

[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.

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.

[The relationship between the resistance of Aegilops umbellulata Zhuk. seedlings to Septoria nodorum Berk. and peroxidase isozyme pattern].

The resistance of Aegilops umbellulata Zhuk. to blotch pathogen Septoria nodorum Berk. has been studied. Cytoplasmic peroxidase activity in normal seedlings could not be used for biochemical prediction of Ae. umbellulata resistance to Septoria blotch. At the same time, isoperoxidases with pI approximately 7.5 and infection-induced activity of anionic isoforms with pI approximately 3.5 were markers of the resistant Aegilops plants. Aegilops resistance to the fungus depended on the activity of cell wall peroxidases.

[Isolation of chitin-specific wheat oxidoreductases].

Anionic peroxidase (IEP approximately 3.5) and oxalate oxidase (IEP approximately 7.0) were isolated from wheat seedlings using chitin. The strength of binding of enzymes with chitin depended on the degree of its acetylation and ionic strength of buffer. It was assumed that the acetyl groups of chitin are involved in sorption of enzymes on this biopolymer. The ability of anionic peroxidase and oxalate oxidase for sorption on chitin allows this biopolymer to be used for isolation of these proteins from plants. Cosorption of anionic peroxidase and oxalate oxidase on chitin suggests that these enzymes cooperate to ensure defensive response of wheat against chitin-containing pathogens.

[Plant resistance inductors and active forms of oxygen. I. The influence of salicylic acid on hydrogen peroxide production in common cultures of wheat callus and bunt pathogen]. / Induktory ustoichivosti rastenii i aktivnye formy kisloroda. I. Vliianie salitsilovoi kisloty na generatsiiu perekisi vodoroda v kletkakh kallusov pshenitsy pri infitsirovanii vozbuditelem tverdoi golovni.

The influence of salicylic acid (SA) on cell resistance to bunt pathogen in wheat calluses has been studied. Cell staining by diaminobenzidin substratum (DAB-cells) was conditioned by hydrogen peroxide (H2O2) generation with the involvement of oxalate oxidase (OO). In the control group, DAB-staining was typical only of rhizoid cells (up to 50%). The infection caused no significant increase in the number of such cells; however, in the zone of fungus penetration parenchyma-like DAB-stained cells were observed (up to 24%). Under the influence of SA, the number of DAB-stained cells did not change, but increased in the zone of fungus penetration (up to 36%). Besides, SA increased OO activity and accelerated proembyogenic complex formation in the calluses, which, unlike rhizoids, were not sensitive to the phytopathogen. The infection caused an increase of OO activity in the cytoplasm and in an intercellular fraction, and an inhibition of an extracellular fraction of the enzyme.

[Plant resistance inductors and active forms of oxygen. II. The influence of chitooligosaccharides on the production of hydrogen peroxide with the involvement of oxalate oxidase in common cultures of wheat calluses and bunt pathogen]. / Induktory ustoichivosti rastenii i aktivnye formy kisloroda. II. Vliianie khitooligosakharidov na produktsiiu perekisi vodoroda s uchastiem oksalatoksidazy v sovmestnykh kul'turakh kallusov pshenitsy i vozbuditelia tverdoi golovni.

We have studied the influence of various concentrations of chitooligosaccharides (CO) on hydrogen peroxide (H2O2) generation, with the involvement of oxalate oxidase (OO), in rhizoids and in zones of hard brand pathogen penetration, as well as on oxalate oxidase activity in wheat calluses. In the control group, diaminobenzidin (DAB)-staining was typical of 30% of peripheral rhizoid cells, which provided their resistance during infection. In the zone of fungus penetration, the appearance of DAB-stained parenchyma-like cells was observed. Simultaneously, the activity OO cytoplasmic fraction increased, whereas OO activity in its ion-bound fraction was suppressed. Low concentrations of medication induced rhizoid formation, increased the number of DAB-stained cells in the pathogen penetration zone, and induced OO activity in cytoplasmic and ionically cell wall-bound fractions. On the contrary, a high concentration of CO (100 mg/ml) suppressed rhizoid formation, Oo activity and the fungus growth. The discovered correlation between enzymatic activation under CO influence, the high level of protective response during infection, and the intensity of rhizoid formation may suggest the community of protective and morphogenetic mechanisms of reactions in plant cells in respect to hydrogen peroxide production.

"Chitin-specific" peroxidases in plants.

The activity of various plant peroxidases and the ability of their individual isoforms to bind chitin was studied. Some increase in peroxidase activity was observed in crude extracts in the presence of chitin. Activated peroxidases of some species fell in the fraction not sorbed on chitin and those of other species can bind chitin. Only anionic isoperoxidases from oat (Avena sativa), rice (Oryza sativa), horseradish (Armoracia rusticana), garden radish (Raphanus sativus var. radicula), peanut (Arachis hypogaea), and tobacco (Nicotiana tabacum Link et Otto) were sorbed on chitin. Both anionic and cationic isoforms from pea (Pisum sativum), galega(Galega orientalis), cucumber (Cucumis sativus), and zucchini (Cucurbita pepo L.) were sorbed on chitin. Peroxidase activation under the influence of chitin was correlated to the processes that occur during hypersensitive reaction and lignification of sites, in which pathogenic fungus penetrates into a plant. The role of chitin-specific isoperoxidases in inhibition of fungal growth and connection of this phenomenon with structural characteristics of isoperoxidases are also discussed.