NITROGEN FIXING BACTERIA OF SPRING WHEAT ROOT ZONE

The paper presents the study of active nitrogen fixation bacteria of genera Azotobacter, Azospirillum, Bacillus, Flavobacterium, Enterobacter and Pseudomonas isolated from root zone of spring wheat plants. The ability of selected diazotrophs to form associative systems with spring wheat was investigated. The most significant increase of molecular nitrogen fixation activity in root zone of plants was observed under the Azospirillum species background.

The problem of biological fixation of atmospheric nitrogen, despite almost a century history, remains one of the major in biological science. In the 70s of the last century the phenomenon of increased nitrogen fixation activity in the root zone of plants, called associative nitrogen fixation was discovered. Studies in this area began with the works of J. Dobereiner [1], where she suggested that the growth and biomass formation of tropical grasses take place owing to atmospheric nitrogen fixation. Many subsequent studies have shown that nitrogen-fixing bacteria easily form associations with various non-legume crops where donor plant provides diazotrophs with available sources of carbon and phosphorus and creates favorable conditions for the nitrogen fixation, and uses, in turn, assimilated nitrogen. Due to the ability to form such associations, diazotrophs adapt better to the environment and have advantages in nutrients supply over other microrganisms.
The ability to establish stable associations of cultivated plants with diazotrophs, analytically selected from agrobiocenoses, is most catching in practical terms and can be considered as the most important measure aimed on the conservation of biological diversity and environmental sustainability.
The aim of this study was to investigate the microbial nitrogen fixation communities of root zone of spring wheat plants and to obtain the culture of diazotrophs that can form active associations with plants.
Materials and methods. Nitrogen fixating bacteria isolated from the inter-row soil, soil washed from rhizosphere and roots of spring wheat plants grown under the small-pot field experiments on shallow black leached light loess loamy soils.
Active diazotrophs were isolated from batch culture after definition of their nitrogenase activity [2]. Batch cultures, which nitrogenase activity was greater than 0.3 mg of nitrogen per 1 ml of culture medium, were cultivated on solid culture media: potato agar, Kaseras, Dobereyner, Eshbi, and agar medium with glycerol.
Green house experiments were conducted in black leached soils with following agrochemical parameters: humus content in the plow layer -3.6%, mobile forms of phosphorus (by Kirsanov) -210-240 mg P 2 O 5 , exchangeable potassium (by Kirsanov) -160-170 mg of K 2 O per 1 kg of soil pHw -6.5. Soil moisture was maintained at 60% of full water capacity. Used pots -12 × 15 cm, capacity -2.0 l. Seeds of spring wheat of Rannya 93 variety were sown to a depth of 2.0 cm, 20 plants per pot. Experiment repetition -10-fold.
Scheme of experiments: 1 -w/o inoculation (control), 2 -inoculation with diazotrophs. Bacterial cultures were grown for three days on the following medium: Pseudomonas -solid medium with glycerol, Azotobacter -Eshbi medium with mannitol, Flavobacterium -Eshbi medium with glucose, Bacillus -potato agar with glucose, Enterobacter -pea agar medium, Azospirillum -potato agar with malate. Presowing seeds treatment was performed with the suspension of diazotrophs with the rate of 200-300 thousand bacterial cells per seed. Experiments Duration -30-40 days after emergence. At the end of this period nitrogen fixation activity was determined by acetylene method in undisturbed soil monolith with roots.
Results and discussion. The results obtained have shown favorable conditions for the development of nitrogen fixing bacteria of different taxonomic and ecologicaltrophic groups in rhizosphere and rhizoplan of spring wheat plants.
More than 1000 diazotroph cultures were analytically selected from different areas of root zone of spring wheat. It was established that only 310 batch cultures (31%) have displayed nitrogenase activity over 0.3 mg of nitrogen per 1 ml of culture medium per day. 60 cultures with high nitrogenase activity were isolated into the pure culture.
Generic distribution of the selected active diazotroph strains is shown on Figure 1. FIGURE. 1. Generic composition of nitrogen fixing associations of root zone of spring wheat plants.
Ability of the selected active diazotroph strains to form effective associations with spring wheat plants was evaluated in green house experiments. As is known from the literature diazotrophic bacteria can not be referred as associative microorganisms based only on their ability to actively fix molecular nitrogen in pure culture in a nutrient medium. It is necessary to ensure its ability to enhance associative nitrogen fixation at introduction into the root zone of plants, since there is no direct relationship between nitrogenase activity in pure culture and in association with plants. Some strains that were able to actively fix nitrogen in pure culture were unable to form effective nitrogen fixing associations with plants and vice versa [3]. This is consistent with some authors stating that the controlling factor in the formation of effective diazotrophic associations with plants and increase of inoculated plants productivity is multifunctional effect of rhizospheric diazotrophs on plants [4], and as a result, improvement of nitrogen nutrition of crops due to the fixation of molecular nitrogen. Figure 2 shows the ability of diazotrophs of Azotobacter genus to form associations with spring wheat plants and enhance nitrogenase activity in the root zone. Data depicted on Figure 2, shows associative nitrogen fixation activity in the root zone of spring wheat plants inoculated with Azotobacter, ranged from 1.7 to 2.3 mg of nitrogen / plant / hour depending on the strain activity. All isolates have promoted increase of this parameter in 2.8 -3.8 times as comparing to the variant without inoculation. It was noted that pure Azotobacter cultures have significantly differed from each other in nitrogen fixing activity (in 1.5 -4.3 times), while the difference in associative nitrogen fixation values at inoculation of spring wheat of Rannya 93 variety with these strains was significantly lower -not exceeding 35%.
Ability of Bacillus genera diazotrophs to form associations with spring wheat and enhance nitrogen-fixing activity in plants root zone is shown on Figure 3. Data presented on Figure 3 shows that only few isolated bacilli strains, capable of fixing molecular nitrogen in pure culture, were able to form active nitrogen fixing association with spring wheat plants. Thus, nitrogenase activity in the root zone of plants inoculated with Bacillus sp. B75, Bacillus sp. B148, Bacillus sp. B206, was at the control plants level. The remaining strains have increased associative nitrogen fixation activity, ranged from 1.4 to 2.3 mg of nitrogen / plant / hour. However, no correlation between the activity of nitrogen fixation in pure culture and in association with spring wheat plants was observed. Bacillus sp. B75 strain has showed maximum nitrogenase activity in pure culture (2.8 mg of nitrogen / 1 ml of culture medium / day), and at the same time, its introduction into the root zone of plants have not influenced on associative nitrogen fixation. Some bacilli strains (Bacillus sp. B78, Bacillus sp. B135, Bacillus sp. B194) possess high nitrogenase activity both in pure culture and in association with of spring wheat plants.
Selected diazotrophs of Pseudomonas genus were able to form active associations with spring wheat (Fig. 4). Results obtained have shown that only four strains of Pseudomonas genus, (Pseudomonas sp P32, Pseudomonas sp P41, Pseudomonas sp Pseudomonas sp P47 and P53) have promoted molecular nitrogen fixation in the root zone of spring wheat plants through the formation of active associations with host plants. Nnitrogenase activity of Pseudomonas -spring wheat systems was on the same level as the associative systems of Azotobacter with spring wheat and spring wheat -Bacillus sp.
Diazotrophs of Flavobacterium genus were unable to enhance nitrogen fixation activity in the plants root zone, i.e., these strains were not able to form active associative systems with the spring wheat (Fig. 5).
Ability of diazotrophs of Enterobacter genus to form associations with spring wheat and enhance nitrogenase activity in the root zone of plants is shown on Fig. 6.
According to the data obtained, out of 9 strains of Enterobacteriaceae, characterized with high nitrogen fixation activity of in pure culture (from 2.2 to 4.7 mg of nitrogen / 1 ml of culture medium / day) only 3 strains (Enterobacter sp. E18, Enterobacter sp . E80 and Enterobacter sp. E388) were not able to increase nitrogenase activity in the root zone of spring wheat plants. The remaining strains were able to form associative system of Enterobacteriaceae with spring wheat, although the activity of this system was low (from 0.9 to 1.4 mg nitrogen / plant / hour) and was lower as comparing to the associative systems of Azotobacter or Bacillus with spring wheat.
Study of ability of Azospirillum bacteria to form associative systems with spring wheat have showed the largest nitrogen fixing activity of these bacteria as compared to others investigated microorganisms. Their introduction into the root system of plants ensures formation of active associations of Azospirillum strains with spring wheat (Fig. 7).