Symbiotic Potential of Bradyrhizobium japonicum Strains with Different Growth Rates.

Aim: To study genetic properties of soybean nodule bacteria with different growth rates, to research the ability of rhizobia to produce phytohormonal substances and estimate their symbiotic potential in the conditions of combined inoculation and mono-inoculation of soybean plants. Methods: Sequencing of the 16S-23S rDNA intergenic spacer of soybean rhizobia was performed on ABI 3130xl Genetic Analyzer. Determination of the qualitative and quantitative composition of phytohormones in culture liquid of Bradyrhizobium japonicum strains was performed by SDTLC-chromatography of high resolution. Symbiotic potential of the strains was estimated in the field experiment in Polissia area of Ukraine. Results: It has been established that strains of soybean nodule bacteria with different growth rates by the structure of ITS region belong to different genetic groups: USDA 6, USDA 110 and USDA 123. The studied rhizobia differ in the levels of extracellular phytohormones synthesis: the slow-growing B. japonicum strain 46 produces more amounts of auxins, but the intensive-growing B. japonicum strain KB11 significantly exceeds in the amounts of cytokinins. It has been shown that an important factor of increasing soybean productivity is the formation of balanced symbiotic systems of the host plant with several complementary strains but serologically and genetically different strains of rhizobia of the same species. Conclusions: Combined inoculation of soybean seeds with two active strains B. japonicum 46 and B. japonicum KB11 allows more fully realizing the potential of symbiotic partners and receiving the higher yield.

[AUXINS AND CYTOKININES SYNTHESIS BY BRADYRHIZOBIUM JAPONICUM UNDER FLAVONOIDS INFLUENCE].

AIM: Research the ability of different by effectiveness symbiotic nitrogen-fixing soybean bacteria Bradyrhizobium japonicum to the synthesis of phytohormones-stimulators auxins and cytokinins for the actions of plant flavonoids genistein and naringenin. METHODS: Extracellular phytohormonal compound isolated from the supernatant culture liquid of the soybean rhizobia by redistribution of phytohormones in two phases solvent immiscible with each other. Auxins and cytokinins were determined by thin layer spectra densitometry chromatography. RESULTS: Shown the ability of symbiotic diastrophic soybean strains to synthesize auxins (4-1067 mg/g of absolutely dry biomass) and cytokinins (141-1554 mg/g of absolutely dry biomass). Cultivation soybean rhizobia in the presence of flavonoid compounds genistein and naringenin leads to the narrowing of the range and reducing the number of phytohormones: unchecked synthesis of indole-3-carboxylic acid, indole-3-carbinol, indole-3-acetic acid hydrazide and zeatin. CONCLUSIONS: Depressing effect of flavonoids on the phytohormones in soybean rhizobia synthesis is probably due to changes in metabolism microsymbiotic bacteria that are not aimed at the synthesis of secondary metabolites and to launch effective nodulating mechanisms, and also the concentration of flavonoid compounds in the nutrient medium.

INFLUENCE OF BACILLUS AMYLOLIQUEFACIENS SUBSP. PLANTARUM IMV B-7404 STRAIN EXOMETABOLITES ON PHENYLALANINE AMMONIA-LYASE ACTIVITY IN WINTER WHEAT SEEDLINGS.

Influence of Bacillus amyloliquefaciens subsp. plantarum IMV B-7404 strain exometabolites on phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) activity in winter wheat seedlings has been studied. A significant increase of PAL activity at 4-6 hours after treatment of plant roots with Bacillus amyloliquefaciens subsp. plantarum IMVB-7404 strain exometabolites and in case of leaves infection with Bipolaris sorokiniana plant pathogen has been shown. It was established that PAL activity changed along with a decrease of the infected surface area of the leaves evidenced for the induction of response in winter wheat seedlings induced by IMV B-7404 strain exometabolites. It was concluded that the studied exponents could be used as model systems in the research of phytoimmunity induction mechanisms.

[SYNTHESIS OF PHYTOHORMONES BY NOCARDIA VACCINII IMV B-7405--PRODUCER OF SURFACTANTS].

AIM: To study the synthesis of phytohormones (auxins, cytokinins, abscisic acid) under cultivation of Nocardia vaccinii IMV B-7405 (surfactants producer) in media containing different carbon sources (glycerol, refined sunflower oil, as well as waste oil after frying potatoes and meat). METHODS: Phytohormones were extracted from supernatants of culture liquid (before or after surfactant separation) by ethylacetate (auxins, abscisic acid) and n-butanol (cytokinins), concentrated and purified by thin-layer chromatography, then quantitative determination was performed using a scanning Sorbfil spectrodensitometer. RESULTS: While growing in medium with refined oil IMV B-7405 strain synthesized 1.8 ± 0.09 g/l extracellular surfactant, also maximum amount of auxins (245-770 µ/l) and cytokinins (134-348 µl). Cultivation of N. vaccini LMV B-7405 on waste oil was accompanied by decreasing amount of phytohormones to 23-84 µ/l (auxins) and 16-90 µ/l (cytokinins) and increasing surfactant concentration to 2.3-2.6 g/l. The level of abscisic acid synthesis was practically not dependent on the nature of growth substrate, was substantially lower than that of auxins and cytokinins and ranged from 2 to 12 µ/l. CONCLUSIONS: Obtained data demonstrate the possibility of using oil-containing industrial waste for the simultaneous synthesis of both surfactants and phytohormones, and indicate the need for studies of the effect of producer cultivation conditions on the biological properties of the target products of microbial synthesis.

[Antagonistic activity of Bacillus amyloliquefaciens subsp. plantarum IMV B-7404 and BIM B-439D strains towards pathogenic bacteria and micromycetes].

In this study the antagonistic activity of strains Bacillus amyloliquefaciens subsp. plantarum IMV B-7404 and BIM B-439D against bacterial and fungal pathogens of agricultural crops has been researched. It is shown that both strains of bacilli demonstrated a high level of antagonism to the vascular bacteriosis pathogen, average level of antagonism to micromycetes--root rot pathogens. To ofiobulez pathogen strain B. amyloliquefaciens subsp. plantarum BIM B-439D was more active. Cultural liquid of this strain effectively inhibited the spore's germination of pathogenic micromycetes Penicillium expansum and Botrytis cinerea. Both strains of bacilli synthesized several hydrolytic exoenzymes: proteases, amylases, ß-glucanases, chitinases and xylanases. The obtained data suggest the possibility of expanding the range of strain B. amyloliquefaciens subsp. plantarum BIM B-439D application for plant protection, as well as the need for further researches of the exometabolites spectrum of strain B. amyloliquefaciens subsp. plantarum IMV B-7404 and their biological activity in order to create an effective bioformulation for crop protection.

[Synthesis of extracellular phythohormones by Bacillus strains isolated from different ecological sources].

Synthesis of extracellular phytohormones by bacteria strains of the genus Bacillus isolated from different ecological sources (soil and plant) has been researched. It has been shown that phytohormone's synthesis ability of the endophytic and free-living Bacillus strains is significantly different as regards the level and spectrum of synthesized compounds. It was concluded that such differences in synthesis of extracellular phytohormones by bacilli are associated with different type of interactions between the studied microorganisms and host plant.

[Role of biocompatible adhesives in the increase of rhizobia physiological activity and productivity of soybean-rhizobia symbiosis].

On the basis of natural exopolysaccharide xanthan and exopolyacrylamide the sticky-gene composition has been developed. Addition of that composition to the culture medium provided a 26.3 times higher viability of Bradyrhizobium japonicum UCM B-6035 cells during its storage. Introduction of plant growth regulators biosil or ivin into this composition increased the survival of rhizobia. Application of gel inoculant B. japonicum favored more intensive growth of rhizosphere microorganisms, nutrient's accumulation in the soil and increased productivity of soybean-Rhizobium symbiosis.

[Activity of main enzymes of ammonium assimilation in Bradyrhizobium japonicum under the influence of plant flavonoid inductors].

The activity of key enzymes of ammonium assimilation in cell-free extracts of soybean rhizobia characterized with different effectiveness in symbiosis, has been researched. It has been found out that at highly efficient strain Bradyrhizobium japonicum UKM B-6035 the most active links in the system of ammonium assimilation reactions are glutamate synthase (glutamate-oxoglutarate-aminotransferase, GOGAT) and glutamate dehydrogenase (GDH). Whereas the most active links at the ineffective strain B. japonicum 21110 are glutamine synthetase (GS) and GDH. Under the influence of specific for soybean-rhizobia symbiosis flavonoid genistein (0.01 nm) GS-activity of the highly efficient strain has considerably increased (60.9 times), and this activity has exceeded the one of the ineffective strain more than twice. GDH has been activated in the presence of genistein in both researched strains. The nonspecific flavonoid naringenin has not stimulated GS-activity of the highly efficient rhizobia, but has stimulated the activity of GDH-way.

[Glutamate dehydrogenase activity of Bradyrhizobium japonicum in the presence of phytoregulators].

Influence of plant growth regulators ivin and emistim C, and flavonoids daidzein and quercetin on the glutamate dehydrogenase activity of soybean nodule bacteria, with contrasting symbiotic properties, were studied. It was shown that all used phytoregulators stimulated glutamate dehydrogenase activity of Bradyrhizobium japonicum 71t (the strain with highly efficient symbiotic properties) 1.2-4.9 times. Bradyrhizobium japonicum 21110 (the strain with inefficient symbiotic properties) diminished the enzyme activity in the presence of all phythoregulators except for ivin.

[Effect of plant growth regulators on physiological activity of Bradyrhizobium japonicum ].

Influence of plant growth regulators Ivin, Emistim C, Eney and Agrostimulin on the biomass production and exopolymers synthesis of soybean nodule bacteria, which have contrasting symbiotic properties, and glutamine synthetase activity of their cell-free extracts were studied. It was shown that the processes of the biomass and exopolymers accumulation had an opposite direction. Of all preparations only Ivin and Agrostimulin intensificol growth activity of the microorganisms under study. The level of glutamine synthetase activity and this enzymatic reaction specificity to the bivalent metal ions were determined by the special features of Bradyrhizobium strains and nature of the plant growth regulators. Only in the presence of Eney the increase of glutamine synthetase activity of both cultures of Bradyrhizobium japonicum was established.