NATURA MONTENEGRINA, Podgorica, 2013, 12(2):

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1 NATURA MONTENEGRINA, Podgorica, 2013, 12(2): ORIGINAL RESEARCH PAPER EFFICIENCY OF MICROBIOLOGICAL PREPARATIONS BASED ON CYANO-RHIZOBIAL ASSOCIATIONS AND NODULE BACTERIA FOR SOYBEAN PRODUCTIVITY Olena PATSKO 1, Yevheniia KONOTOP 1, Nadiya VOROBEY 2, Sergiy K O T S 2 and Nataliya TARAN 1 1 Educational and Scientific Centre Institute of Biology, Taras Shevchenko National University of Kyiv, 64/13 Volodymyrska Str, Kyiv, Ukraine, patsko_lena@ukr.net, golovatyuk.yevgeniya@gmail.com 2 Institute of Plant Physiology and Genetics of NASU, 31/17 Vasylkivska Str., Kyiv, Ukraine, SYNOPSIS Key words: soybean, cyano-rhizobial associations, Rhizogumin, Tn5-mutant strains, productivity. In present study efficiency of pre-sowing treatment of soybean (Glycіne max (L.) Merr.) seeds by manufactured fertilizer Rhizogumin, based on bacterial suspension of soybean nodule bacteria Bradyrhizobium japonicum 46 and M-8, and inoculation with cyano-rhizobial associations, based on the nodule bacteria, their Tn5 mutants and the cyanobacteria Nostoc PTV for plant productivity and resistance to environment was investigated. Obtained data on the influence of complex bacterization for soybean growth, development, quantitative/qualitative indexes of productivity under legume-rhizobial symbiotic conditions indicate the prospects of bacterial preparations based on Bradyrhizobium japonicum 46 and M-8 in manufactured fertilizer Rhizogumin and cyanobacteriarhizobia associations, including their Tn5-mutant strains. INTRODUCTION Today one of the important fundamental problems of agriculture is the optimization of plant nutrition in a transformed environment. The problem of modern technological burden on agroecosystems requires exploration and development activities that would promote the development of adaptive agricultural land and, thus, obtaining environmentally friendly products. Molecular nitrogen fixation by symbiotic systems of plants and nodule bacteria is the process of planetary importance, which is of particular relevance in the implementation of a highly productive and environmentally friendly farming. In most countries, leguminous plants have a key role in overcoming the problems associated 539

2 Natura Montenegrina 12(2), 2013 with the energy crisis, lack of protein, and the need to protect the environment. In addressing these issues from the viewpoint of fundamental science the interaction between prokaryotes and higher plants, the structure forms of fixed nitrogen, its metabolism, the role of genetic and environmental factors attract special attention. The ability of a plant to supply all or part of its N (nitrogen) requirements from biological nitrogen fixation (BNF) in its roots can be a great competitive advantage over non-n 2 -fixing neighbors. BNF is the conversion of atmospheric N 2 to ammonium, a form of N that can be utilized by plants. However, BNF is in the sole domain of certain bacteria (diazotrophs), which contains nitrogenase, the enzyme complex that catalyzes the conversion of N from the gaseous to the combined form. Occurrence of N 2 -fixing bacteria with higher plants is not uncommon, but in most cases these are only associations, in which relatively free-living bacteria grow in the rhizosphere, on the rhizoplane, or more rarely, in non-specialized intercellular spaces in plants (Vessey, 2003). The transfer of fixed N from the bacterium to the plant in these associations is relatively low, and the relationship between the two organisms could be viewed as opportunistic rather than mutualistic. However, in a much smaller proportion of cases across the plant world, the association between plant and bacterium is much more intimate, with the N 2 -fixing bacterium being housed within specialized plant organs. In these truly mutualistic symbioses, the genetics and physiology of the plant and bacteria are integrated to the extent that the two organisms can appear to function nearly as one. On several occasions, symbioses have evolved between terrestrial plants and N 2 - fixing bacteria, leading to the existence of specialized organs on the host plants that provide excellent environment for the prokaryotes to infect, live, and fix N 2. Although the genetic backgrounds and physiological functioning of these symbioses can be seen as very diverse, there are several developmental and physiological imperatives that must be met for successful symbioses between host plants and their N 2 -fixing microsymbionts. These imperatives include: the ability of the microsymbiont to infect and colonize host plant organs, the ability of the host plant to supply energy and nutrients to the microsymbiont, the ability of the host plant and microsymbiont to regulate O 2 flux, the ability to transfer the fixed N from the microsymbiont to the host. Each symbiosis can be admired for the elegant means by which the host plant and microsymbiont integrate to form the mutualistic relationships so important to the functioning of the biosphere (Vessey, 2004). Today cyanobacteria are very interesting and perspective subject of the biotechnology that positively affect on soil fertility and activity of soil biota. Nitrogenfixing cyanobacteria, particularly Nostoc, form symbioses with plants ranging from algae to angiosperms and provide fixed N to the host (Rai et al., 2000). Nitrogenfixing cyanobacteria also occur epiphytically (phyllosphere and/or rhizosphere) on plants growing in aquatic and high-humidity environments (Freiberg, 1999; Whitton & Potts, 2000). The creation of facultative associations involving 540

3 Patsko et al.: EFFICIENCY OF MICROBIOLOGICAL PREPARATIONS BASED ON... cyanobacteria located extracellularly on or in the plant seems to be more promising as an alternative. With their ability to colonise a wide range of plants and plant tissues or organs, their capacity for aerobic N 2 fixation, and their flexible modes of C and N nutrition, diazotrophic cyanobacteria are a more likely candidate than rhizobia to form productive associations with plants. Nostoc is the most common cyanobiont in natural symbioses and, therefore, the ideal choice. Unicellular forms, by contrast, are rarely found as N 2 -fixing partners in natural symbioses. Attempts to create artificial symbioses using unicellular forms, as well as attempts to create intracellular symbioses, have not been successful (Rai et al., 2000). However, cocultivation of plant seedlings (e.g. from wheat, corn, sugar beet, rice and marine mangroves) and N 2 -fixing cyanobacteria (particularly Nostoc) in liquid and sand cultures, can lead to colonisation of the roots and support seedling growth (Toledo et al., 1995; Rai et al., 2000). Nostoc has the widest range of habitats, hosts and locations in host tissues (extra- or intracellular). It is an aerobic N 2 -fixer with versatile modes of C nutrition (phototrophic, photoheterotrophic, heterotrophic). All the biochemical and developmental requirements of N 2 - fixation are extant within the cyanobacterial cells, and their location within the plant cell is not essential. From those competent to colonise, further selections and modifications can be made for higher rates of N 2 -fixation, herbicide resistance, N 2- fixation in the presence of combined N, and ammonia release. A new step towards enhancement of plant productivity by biological products is up-to-date knowledge about the effectiveness of nodule bacteria (Bradyrhizobium japonicum) and manufactured fertilizer based on it. Besides the use of cyanobacteria Nostoc for the formation of rhizobium-legume consortium is effective for its functioning. Creation the bacterial preparations on their basis are promising for improving plant productivity. That is why the use of biological agents based on nitrogen-fixing microorganisms, including rhizobia, is one of the main methods for improving plant productivity, which allows maintaining soil fertility and the ecological balance of the environment. Use of manufactured fertilizers, based on the nodule bacteria, and, on the other hand, creation and selection of compatible cyano-rhizobial associations, is one of the ways of biological stimulation of legume-rhizobial symbiosis, which increases the importance of the rhizobia-plant interaction and efficiency of bacterial preparations. MATERIALS AND METHODS Soybean, Glycine max (L.) Merr., cultivar Maryana was used in experiments. For inoculation of seeds slow-growing bacterium Bradyrhizobium japonicum - Tn5-541

4 Natura Montenegrina 12(2), 2013 mutant 646 strain of B. japonicum - T66 (nitrogen-fixing microorganisms from the collection of the Institute of Plant Physiology and Genetics NAS of Ukraine) was involved. These bacteria were obtained by transposon mutagenesis as a result of conjugation with Escherichia coli (psup2021: Tn5) (Reznikoff, 2003) and selected for improved symbiotic properties from other genetically-altered nodule bacteria. Cyanobacteria Nostoc PTV from the collection of the Institute of Hydrobiology National Academy of Sciences of Ukraine, which was grown until stationary phase of growth on Fitzgerald nutrient medium in modified Zehnder and Gorem (N 11) (Svircev, 1997), was used for creation a binary compositions of nitrogen-fixation culture of microorganisms. Research was conducted in laboratory condition and in model vegetative experiments. In laboratory experiments the soybean seeds germinated in Petri dishes and determined indexes such as germinative energy, seed germination, the length and mass of formed shoots (Loboda et al., 1991). In model vegetative experiments each 6 plants were grown in 15 kg Wagner containers, which were previously sterilized by 20% solution of H 2 O 2. Washed river sand with Helrigel nutrient mixture was used as a substrate. Before sowing seeds were surface sterilized in 70% ethanol for 15 min. Then, depending on the mode of experiment seeds were performed for 60 min inoculation with trained suspensions of related strains B. japonicum, cyanobacteria N. PTV and binary compositions of these strains of diazotrophs. Study of nitrogen fixing activity data symbiotic system determined by the Hardy method (Hardy et al., 1968). The gas mixture was analyzed by gas chromatography Agilent Technologies 6855 Network GC System (USA). The assessment was performed in five-fold repetition. The content of photosynthetic pigments in leaves of soybean plants determined by the Wellburn methods (Wellburn, 1994) on the spectrophotometer Smart Spec Plus (Biorad, USA). The assessment was performed in three-fold repetition. Sampling the plants for study the level of nitrogen-fixing activity and the content of photosynthetic pigments was performed in the budding phase of development. Study of protein content in soybean seeds was performed by Lowry method (Lowry et al., 1951). Plant protein extraction (500 mg) was performed with hot ethanol. The concentration of protein in the test samples was determined by using gauge design, built with serum albumin. The quantity and qualitative composition of amino acids in soybean seeds was determined by ion exchange liquid chromatography with the use of automatic analyzer T339 (Czech) on the basis of ninhydrin detection method (Zubay, 1998). Efficiency of pre-sowing treatment of soybean seeds by manufactured fertilizer Rhizogumin was tested in a field with further condition: black soil with humus 542

5 Patsko et al.: EFFICIENCY OF MICROBIOLOGICAL PREPARATIONS BASED ON... content in the soil plow layer 4,38-4,53%, ph of salt extraction 6,9-7,3, content of nitrogen - 0,27-0,31%, phosphorus 0,15-0,25% and potassium - 2,3-2,5%. Rhizogumin (produced by Institute of Agricultural Microbiology and Agricultural Production, National Academy of Agrarian Science of Ukraine) is fertilizer based on bacterial suspension of soybean nodule bacteria Bradyrhizobium japonicum 46 and M-8 with bacteria titer of at least 1 billion cells /g. Physiological responses of soybean to the effect of experimental conditions were assessed by the primary non-specific reactions on the developmental stages of blooming and grain filling. The intensity of lipid peroxidation (LP) was estimated by accumulation of malonic dialdehyde (MDA) (Heath, Packer, 1968). Photosynthetic pigments were extracted from leaves with pre-chilled 80% acetone, further the pigment content was determined spectrophotometrically (Arnon, 1949). Nutritional value of soybean seeds was evaluated for protein content (Lowry et al., 1951) and oils. Crude oils were extracted with n-hexane. Oil percentages were determined by weight difference according to control. Each experiment was performed at least three times. The data were subjected to analysis of variance (ANOVA) with subsequent Student s t-test (P 0,05). Data on the diagrams are expressed as the means of replicates ± standard deviation. RESULTS AND DISCUSSION One of the criteria of assessing the effectiveness and feasibility application of bacterial fertilizer is the degree of their influence on the plants on the early stages of development. It is known that metabolites of microorganisms, including cyanobacteria may play role of biological stimulants of plant growth and development. Our studies show that treatment of seeds of soybean by monocultures of rhizobia, cyanobacteria and their binary mixtures leads to increasing of the index of germinative energy and seed germination compared with the control without inoculation (Fig. 1). Thus, the index of germinative energy for seeds treatment by Nostoc PTV was 66% (which is 7% higher than control). Especially noticeable stimulation effect on seed germination (increasing by 18,9 %) showed seeds inoculation by binary compositions of Tn5- mutant strain T66 + N. PTV. Of particular note is the stimulating effect of N. PTV and its binary mixtures with nodule bacteria at length of formed seedling. Thus, length of seedlings under the influence of seeds inoculation by N. PTV appeared to be 21% higher than the control (Fig. 2A). Under the monoinoculation by T66 strains of rhizobia, this value remained at control level, whereas binary inoculation by T66 with N. PTV led to its growth by 21,4%.The most effective influence on the rate of mass of seedlings was observed under monoinoculation of seeds by N. PTV. This index increased by 23% 543

6 Natura Montenegrina 12(2), 2013 compared to the control. The binary inoculation by Tn-5 mutants T66 caused increasing seedlings weight by 23.2% compared to monoinoculation of seeds by only nodule bacteria (Fig. 2B). Figure 1: Germinative energy (A) and seed germination (B) of soybean Glycine max (L.) Merr. treated with mono and binary inoculation of cyano-rhizobial compositions of microorganisms: 1 control (soybean seeds germinated without inoculation), 2 pre-sowing monoinoculation of soybean seeds by cyanobacteria Nostoc PTV, 3 а pre-sowing inoculation of soybean seeds by Tn5- mutant strain T66 Bradyrhizobium japonicum, 3b pre-sowing binary inoculation of soybean seeds by Tn5- mutant strain T66 Bradyrhizobium japonicum + Nostoc PTV. Figure 2: Length (A) and mass (B) of seedlings of soybean Glycine max (L.) Merr. treated with mono and binary inoculation by cyano-rhizobial compositions of microorganisms: 1 control (soybean seedlings germinated without inoculation), 2 pre-sowing monoinoculation of soybean seeds by cyanobacteria Nostoc PTV, 3а pre-sowing inoculation of soybean seeds by Tn5- mutant strain T66 Bradyrhizobium japonicum, 3b pre-sowing binary inoculation of soybean seeds by Tn5- mutant strain T66 Bradyrhizobium japonicum + Nostoc PTV. 544

7 Patsko et al.: EFFICIENCY OF MICROBIOLOGICAL PREPARATIONS BASED ON... It is known that cyanobacteria synthesize gibberellins, cytokinins, auxins, vitamins and other important compounds. Credibly, activation of metabolic processes in soybean seeds under the influence of biologically active substances of cyanobacteria led to increasing of seedling length of those inoculated by compositions of microorganisms compared to plants treated only by monocultures of rhizobia. Figure 3: Nitrogen-fixation activity (A) and mass of root nodules (B) of symbiotic consortiums of soybean Glycine max (L.) Merr. formed after pre-sowing mono- and binary inoculation of seeds by cyano-rhizobial compositions of microorganisms: 1 presowing inoculation of soybean seeds by Tn5-mutant strain T66 Bradyrhizobium japonicum, 2 pre-sowing binary inoculation of soybean seeds by Tn5- mutant strain T66 Bradyrhizobium japonicum + Nostoc PTV. The optimal selection of inoculating partners, especially new cyano-bacterial composition based on Tn-mutant nodule bacteria and cyanobacteria, had positive effect on indicator of nitrogen-fixation activity, significantly increasing it and thus improved nitrogen nutrition of leguminous plants and enhance their productivity. In our case, the inclusion of cyanobacteria N. PTV in suspension for inoculation with nodule bacteria resulted in insignificant effect on the change of this indicator (Fig. 3A, B). Significant difference between the nitrogen-fixation activity and weight of root nodules in variants of mono-and binary inoculation was noted. It is known that the effectiveness of different strains of rhizobia and their associations with other organisms through the nitrogen status of plants affects the full development and subsequent functioning of the photosynthetic apparatus. It is shown that the use of studied microorganisms and their association for inoculation of seeds resulted in positive changes in the dynamics of photosynthetic pigments accumulation in leaves of soybean compared with control (Fig. 4A, B). The content of chlorophyll in leaves is directly proportional to the intensity of nitrogen fixation and depends on symbiotic properties of rhizobia. 545

8 Natura Montenegrina 12(2), 2013 Figure 4: С ontent of chlorophylls (A) and carotenoids (B) in soybean Glycine max (L.) Merr. leaves grown after mono and binary inoculation of seeds by cyano-rhizobial compositions of microorganisms: 1 control (soybean grown without inoculation), 2 pre-sowing monoinoculation of soybean seeds by cyanobacteria Nostoc PTV, 3а presowing inoculation of soybean seeds by Tn5- mutant strain T66 Bradyrhizobium japonicum, 3b pre-sowing binary inoculation of soybean seeds by Tn5- mutant strain T66 Bradyrhizobium japonicum + Nostoc PTV. Inoculation of soybean seeds by active straine Tn5-mutant T66, as well as cyanobacteria N. PTV led to an increase of content of chlorophyll a and b in the leaves compared to the control without inoculation. Application of cyano-rhyzobia compositions based on these microorganisms further increased the content of photosynthetic pigments (including carotenoids) as compared to the control without inoculation and compared with options inoculated by monocultures T66 (however, this allowance was minor comparing to variation of monorhyzobia inoculation). In our case, the increase of number of these pigments in leaves under the inoculation of soybean by binary compositions of diazotrophs can serve as a proof of the ability of cyanobacteria affect the metabolism of rhizobia that appears on their symbiotic properties. Symbiotrophic form of N has a positive impact on the full development and subsequent functioning of the photosynthetic apparatus of plants and promotes the accumulation of organic matter and, consequently, increases the yield of plants as the basis for biological productivity of the plant organism. The results of our research showed that application of cyano-rhizobial compositions for seed inoculation did not lead to significant changes of grain productivity of plants that is believed to be an integral index of interaction and efficiency of symbiotic partners. However, crops in variant of inoculated seeds with monoculture of researched strain of bacteria and its composition with N. PTV were more effective and even exceeded non-treated control by 11,9 and 39,6% (tab. 1). 546

9 Patsko et al.: EFFICIENCY OF MICROBIOLOGICAL PREPARATIONS BASED ON... Table 1: Grain production and protein content in soybean seeds, inoculation N. PTV, strains of B. japonicum T66 and binary compositions based on them. Variant Control without inoculation Crop g / conteiners Quantity of grain in crop compared to control (without Protein content in seeds,% 25,2 ± 0,4-30,2 Nostoc PTV 28,2 ± 0,8 11,9 33,8 Strain Т66 35,6 ± 0,4 41,2 39,2 % to mono inoculation Strain Т66 + N. PTV 33,0 ± 0,8 31,0 43,2 110,2 It is known that qualitative measure of effectiveness of rhizobium-legumesymbiosis is the protein content in seeds or in leaves that confirms the determination of establishing symbiotic relationship between plant and nodule bacteria (Plazinsky, 1997). Total protein content in our experimental variants with mono- and binary inoculation rose compared to control. Although no significant difference in terms of grain productivity of soybeans processed with mono- and binary cultures of microorganisms were admitted. The most effective options for seed inoculation was binary composition T66 + N.PTV, that resulted in higher protein content in soybean seeds by 10,2% compared with the corresponding variations after monorhizobium processing. The main criterion of biological value of proteins is their amino acid composition. It has been shown that bacterial mono- and binary inoculation positively influenced the quantity and quality content of amino acids. Compared with the control treatment of soybean seeds by nitrogen-fixing microorganisms led to increasing of both total amino acid content (Fig. 5) and content of each of them separately. Significant difference in the total amino acid content between mono- and binary inoculation was not recorded. However, lysine content that is known to be the most limited essential amino acid, in variant of binary cyano-rhizobium inoculation increased in comparison with the control and its maximum reached 34,5% in the variant T66 + N. PTV. The highest content of one of amino acids that limit the rate of protein synthesis was also observed in the binary version of inoculation: content of histidine increased by 21,4% for the treatment by T66 + N. PTV. However, estimating of the total content of essential and replacement amino acid (Fig. 5) showed tendency to increase of the number of essential amino acids in seeds in variant of binary cyano-rhizobium inoculation (options 3b) compared with control. In the soybean seeds the amount of replacement amino acid also increased, which confirms the effectiveness of binary compositions for balancing amino acid composition of this plant. Experiment design in field condition was dedicated to investigation the presowing treatment of soybean seeds by popular on Ukrainian market microbiological 547

10 Natura Montenegrina 12(2), 2013 fertilizer Rhizogumin. It has been shown that pre-sowing treatment of soybean seeds by microbial agents, for example Rhizogumin, as in the absence of rhizobia in the soil as well as in case of their dense population; improves the efficiency of soybeanrhizobia interaction (Volkogon et al., 2005). Figure 5: Total content of essential and replacement amino acids in soybean seeds grown after pre-sowing mono and binary inoculation by cyano-rhizobial compositions of microorganisms: 1 control (soybean grown without inoculation), 2 pre-sowing monoinoculation of soybean seeds by cyanobacteria Nostoc PTV, 3а pre-sowing inoculation of soybean seeds by Tn5- mutant strain T66 Bradyrhizobium japonicum, 3b pre-sowing binary inoculation of soybean seeds by Tn5- mutant strain T66 Bradyrhizobium japonicum + Nostoc PTV. Figure 6: Malonic dialdehyde content in leaves of soybean on developmental stages of blooming and grain filling in control condition and after presowing seed treatment with Rhizogumin. Results of field experiment showed intensification of LP - the initial nonspecific response to stress factor, which revealed in the growth of MDA content in photosynthetic tissues of experimental option with Rhizogumin treatment in a phase 548

11 Patsko et al.: EFFICIENCY OF MICROBIOLOGICAL PREPARATIONS BASED ON... of blooming (Fig. 6). In a phase of grain filling significant decreasing of MDA content in leaves of plants with pre-sowing treatment by Rhizogumin was observed. However, no difference in level of LP between plants on different developmental stages was noticed in a control option. Dose-dependent tendency to increasing of the content of photosynthetic pigments on the critical stage of plant development (Fig. 7), such as a phase of blooming, was observed in plants without pre-sowing treatment, that confirms the importance of N for the synthesis of chlorophyll and other organic molecules with porphyrin ring. In the leaves of soybean that were subject to pre-sowing treatment with microbiological agent increase of chlorophyll a content by 64% was observed. Contents of chlorophyll b and carotenoids were at control values. It is known that in the phase of grain filling redistribution of assimilates with the intense outflow to seeds occurs, which affected the reduction of photosynthetic pigments. Moreover, Rhizogumin using resulted in decreasing of the content of photosynthetic pigments on the stage of grain filling in comparison with the control plants without any treatment. The nutritional value of soybean seeds was estimated by protein and oil content (Fig. 8 A, B). Oil and protein content in seeds increased by 40 and 35% accordingly in plants that were cultivated after pre-sowing treatment by Rhizogumin. That could be assumed as a result of more efficient use of N in the metabolic reactions of plants subjected to pre-sowing treatment by microbiological agent. Fig. 7. Content of photosynthetic pigments in leaves of soybean on developmental stages of blooming and grain filling in control condition and after pre-sowing seed treatment with Rhizogumin. 549

12 Natura Montenegrina 12(2), 2013 Figure 8: Content of crude oil (A) and protein (B) in seeds of soybean in control condition and after treatment with Rhizogumin. CONCLUSIONS Obtained data suggest a positive effect of bacterial mono- and binary inoculation on the nutritional value of soybean seeds. Results of presented complex research will contribute to improving technology of soybean cultivation with the aim to enhance total plant productivity and particularly to promote protein and oil content increasing in seeds that would be useful for food industry. The rational sustainable use of biological fertilizers separately and in combination with other farming practices can allow significantly reducing the chemical pressure on the ecosystem and greatly improving the quality of agricultural products. REFERENCES: ARNON, D.I. 1949: Copper enzymes in isolated chloroplasts: polyphenol oxidase in Beta vulgaris. - Plant Physiology, 24: FREIBERG, E. 1999: Influence of microclimate on the occurrence of cyanobacteria in the phyllosphere in a remontane forest of Costa Rica. - Plant Biology, 1: HARDY, R., HOLSTEN, R., JACKSON, E., BURNS, R. 1968: The acetylene ethylene assay for N 2 -fixation: laboratory and field evaluation. - Plant Physiology, 43:

13 Patsko et al.: EFFICIENCY OF MICROBIOLOGICAL PREPARATIONS BASED ON... HEATH, R. L., PACKER, L. 1968: Photooxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. - Archives of Biochemistry and Biophysics, 125: LOBODA, N., VESNA, B., SYROTA, M. 1991: Handbook on Seed. - Urojay press, Kiev, 352 pp. LOWRY, O., ROSEBROUGH, N., FARR, A., RANDALL, R. 1951: Protein measurement with folin fenol reagent. Biological Chemical, 192: PLAZINSKY, J. 1997: Nitrogen metabolism of the symbiotic systems of cyanobacteria. Cyanobacterial N 2 Metabolism and Environmental Biotechnology. - Narosa Publ. House, 121: RAI, A., SODERBACK, E., BERGMAN, B. 2000: Tansley Review: cyanobacterium plant symbioses. - New Phytologist, 147: REZNIKOFF, W. 2003: Tn5 as a model for understanding DNA transposition. - Molecular Microbiology, 47: SVIRCEV, Z. 1997: Co-cultivation of N 2 -fixing cyanobacteria and some agriculturally important plants in liquid and sand cultures. - Applied Soil Ecology, 6: TOLEDO, G., BASHAN, Y., SOELDNER, A. 1995: In vitro colonization and increase in nitrogen fixation of seedling roots of black mangrove inoculated by a filamentous cyanobacterium. - Canadian Journal of Microbiology, 41: VESSEY, J. 2003: Plant growth promoting rhizobacteria as biofertilizers. - Plant and Soil, 255: VESSEY, J. 2004: Root-based N 2 -fixing symbioses: Legumes, actinorhizal plants, Parasponia sp. and cycads. - Plant and Soil, 266: VOLKOGON, V., SHTANKO, N., SALNYK V. 2005: Efficiency of new biological agent rhizogumin for soybean. Selekciya I nasinnyctvo, 90: WELLBURN, A. 1994: The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolutions. - Plant Physiology, 144: WHITTON, B., POTTS, M. (eds) 2000: The ecology of cyanobacteria. Dordrecht, Kluwer, 760 pp. ZUBAY, G. 1998: Biochemistry. - The McGraw-Hill Companies Inc., 920 pp. Received: 25 July

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