Сельскохозяйственная биология, 2011, № 4, с. 90-95.

УДК 635.65:631.5:631.46

APPLICATION OF MIXED BACTERIAL CULTURES FOR INCREASING YIELD OF LEGUMINOUS PLANTS

V.P. Shabayev

Combined inoculation with mixed cultures of nodule bacteria, Rhizobiuт lеguтinоsarum bv. viceae 250а, Bradyrhizobiuт japonicum 110, R. meliloti ВКМВ117, R. trifolii 348а, and plant growth-promoting rhizobacteria of the genus Pseudomonas, Р. fluorescens 20 or Р. fluorescens 21, was shown to enhance yield of pea and soybean grain and green mass of alfalfa and clove in microfield and pot experiments in condition of Moscow region. Mixed bacterial cultures stimulate symbiotic N₂ fixation and increase the amount of biological N in plants and macro- and micronutrients uptake by yield from the soil. The efficiency of combined inoculation with bacteria depends on application of N and P fertilizers.

Keywords: legumes, nodule bacteria, Pseudomonas, yield, nutrients uptake, N₂-fixation, NPK-fertilizers.

In recent decades, mixed microbial cultures are used to stimulate growth and increase yields of crops (including legumes); these microorganisms have high nitrogen-fixing activity, resistance to environmental impact and competitiveness to the natural microflora (1, 2).
Previously, the authors have established in Pseudomonas fluorescens 20 and P. fluorescens 21 the colonization in root systems and survival in rizoplane of different crops (soybean, winter wheat and fodder beet) (3, 5, 8). P. fluorescens 20 was found to stimulate growth and better yields of cereals (5, 6), root crops (7-9) and spring rape (10).
The purpose of this work - to study the effects of the combined use of nodule bacteria and rhizospheral growth-enhancing bacteria the genus Pseudomonas on productivity and symbiotic nitrogen fixation in different species of legumes.
Technique. The studies were carried out in 1997-2006 on pea (cv Smaragd), soybean (cv Mageva), alfalfa (cv Slavyanskaya mestnaya) and clover (cv VIK 1).
Microfield tests were performed in the south of Moscow province.  11 plants of each crop were grown in vessels without a bottom (0,33Í0,33Í0,33 m;  0,1 m2) that were dug into the top layer of soil (gray forest soil, 30 kg)..
Variant I: the sown seeds were placed on the soil surface and inoculated with water suspensions of nodule bacteria (106-107 cells per plant): peas - with Rhizobium legutinosarum bv. viceae 250A, soy - Bradyrhizobium japonicum 110, alfalfa - R. meliloti ВКМВ117, clover - R. trifolii 348A. The strains of nodule bacteria R. legutinosarum bv. viceae 250A and R. trifolii 348A were obtained from the All-Russia Research and Development Institute for Agricultural Microbiology (St.Petersburg-Pushkin), B. japonicum 110 - from the Institute of Biochemistry and Physiology of Microorganisms (Pushchino, Moscow province), R. meliloti BKMB117 - from the All-Russian Collection of Microorganisms (Moscow).
Variant II: the seeds of each plant were inoculated with the nodule bacteria jointly with Pseudomonads (P. fluorescens 20 and P. fluorescens 21 isolated from the rhizosphere of oats) at the ratio 5:1 that had been earlier proved as the most efficient (3, 4). In addition, there were other versions: for soybean – the variant without inoculation (using autoclaved bacterial cultures); for peas, the control not fixing nitrogen was oats cv Gambo grown under the same conditions without inoculation (using the autoclaved cells) and one inoculated with the strain P. fluorescens 20.
In all experiments, before planting the crops, a nitrogen fertilizer (ammonium nitrate) was introduced into soil at a dose of 0,4 g N/pot (based on 4 g N/m²) against the background of a phosphorous-potassium fertilizer (potassium phosphate monobasic and sulphate) at doses of 0,8 g AI/vessel (based on 8 g AI/m²) except the 2nd variant with clover (only phosphorus- potassium fertilizer). In experiments with soybeans and peas, the 15N-labeled nitrogen fertilizer was used in all the variants in order to estimate the ratio of symbiotically fixed and mineral nitrogen in plant nutrition.
Plants of peas and soybeans were grown to full maturity, alfalfa and clover - during two growing seasons (the 1st year: at flowering stage, alfalfa was harvested three times and clover – once, on the 2nd year both crops were mowed twice).
The proportion of fixed atmospheric nitrogen and mineral nitrogen in peas were established by comparing with non-legumes crops (oats), in soybean – relative to the non-inoculated control (11). The contents of nitrogen and mineral elements in green phytomass were determined (5-10). Along with it, nitrogen isotopic composition was analyzed in peas, soybeans, oats (избыток ат. % 15N) (the excess of atm.% 15N) (3, 6, 8). Performance of experiments – 4-fold.
In the vegetation test, three plants of soybean were grown to early fructification phase (for 85 days till the beginning of formation beans) in pots (9 cm diameter, 30 cm height) filled with a mixture of gray forest soil and sand (1:1 by volume). In this experiment, a phosphorus fertilizer (potassium phosphate monobasic) was introduced into soil at the rate of 0; 5 and 20 mg P2O5 against the background of nitrogen-potassium fertilizers (ammonium sulfate and potassium sulfate - respectively, 4 mg N and 20 mg K2O per 100 g substrate). In one series of the experiment, the seeds were inoculated at sowing with a pure culture of the nodule bacteria B. japonicum 110, in another series – with a mixed culture B. japonicum 110 + P. fluorescens 21. The effects of P. fluorescens 21 were determined – weight of plans, the development of root nodules and their nitrogen-fixing activity (by acetylene method) (1). Performance of experiments – 5-fold.
The obtained data on yields and results of chemical analyses of plants were statistically processed by the dispersion analysis using Statgrafics software.
Results. The inoculation of sowing seeds with the mixed bacterial inoculum significantly increased yields of legumes (Table 1). The combined use of R. legutinosarum bv. viceae 250A and P. fluorescens 20 provided the 20% higher weight of peas grain yield compared to the variant with the pure culture of nodule bacteria. The combined treatment with B. japonicum 110 + Pseudomopas had a positive impact on growth of soybean plants even against the high efficiency of using only nodule bacteria, which was accompanied by more than twice higher grain yield (high responsiveness was manifested owing to the absence of these rhizobia in gray forest soil). In the variant with joint inoculation of soybean with nodule bacteria and P. fluorescens 20 or P. fluorescens 21, the increase in weight of grain yield continued to rise and amounted to 18-21% relative to the variant when using only B. japonicum 110.
The herbal legumes showed the positive effect of using mixed bacterial cultures as well (Table 1). In alfalfa treated with P. fluorescens 20, there was observed 33% higher green mass (in total for two growing seasons). However, in clover, inoculation with both mixed bacterial cultures against the background of the nitrogen fertilizer (as a part of NPK-fertilizers) was ineffective in contrast to other legumes (the1st microfield experiment). The positive effect of the mixed inoculum R. trifolii 348A + P. fluorescens 20 was manifested only against the PK-fertilizer (2nd microfield experiment). In this case, the gain in green mass of clover reached 16% (in total for 2 years).

The combined inoculation with nodule rhizobia and Pseudomonas significantly increased the nitrogen accumulation in legumes. Thus, using P. fluorescens 20 + P. fluorescens 21 on soybean increased the transfer of this element into grain by 8-14%. In total for 3-times harvesting, seed treatment with P. fluoresceps 20  provided in alfalfa the 1,5-fold rise of nitrogen transfer into a green mass during the 1st growing season, and by 26% - in clover during 2 years of experiment.
In the variant of soybean inoculated with the mixed culture, there was observed significantly higher accumulation in the grain of almost all analyzed mineral elements - for some of them the transfer into grain increased by 50% (Table 2). The exception was Co in the variant with P. fluorescens 21: the transfer of this element into grain remained unchanged. In alfalfa, inoculation with the nodule bacteria + P. fluorescens 20 resulted in increased transfer into the green mass of P, Ca, Zn, Cu, Mo (in 1,5 – 1,7 times), K, Mg, Fe and Co (in 1,3-1,4 times) in the 1st growing season. In the 2nd experiment (the mixed bacterial culture R. trifolii 348A + P. fluorescens 20 against the background of PK-fertilizer), clover plants demonstrated enhanced accumulation of mineral elements in a green mass. In total for 2 years, it amounted from 14 to 30%, along with the highest contents of K, Zn and Mo transferred into a harvest (though no change for Cu). Therefore, introduction of bacteria the genus Pseudomonas in soil improves mineral nutrition of legumes.

It should be noted that the observed increase in amounts of nutrients transferred into harvested parts of legumes occurred owing to increase of their yields (relative contents of most elements in plants remained almost unchanged). Chemical elements are known to provide the direct action in plants by entering the active sites of enzymes and the indirect action by affecting metabolic processes. Improved mineral nutrition, undoubtedly, stimulates physiological processes in plants. Particularly, the enhanced photosynthesis (the intensity of CO₂ gas exchange) in soybean inoculated with Pseudomonas was established (3). The increased transfer of mineral nutrients from soil indicated their better availability to plants inoculated with Pseudomonas – such as Mo, which is a part of the nitrogen-fixing enzyme nitrogenase, as well as other elements involved in microbial fixation of molecular nitrogen.
In experiments with peas and soybeans using 15N-labeled nitrogen fertilizers, the starting dose allowed to measure the ratio of symbiotrophic and mineral nitrogen nutrition in these plants (Table 3). Inoculation with P. fluorescens 20 contributed to the increase in contents of symbiotically fixed atmospheric nitrogen (biological nitrogen) in peas grain in1,5 times and the nitrogen from fertilizer and soil - by 14 and 18%, resp. Inoculation of soybean with nodule bacteria + Pseudomonas contributed to 9-13% higher amount of fixed nitrogen in yield along with the enhanced assimilation of nitrogen from soil (by 14-29% higher) and the labeled fertilizer (by 8-15%).

In the vegetation test on soybean, rhizospheral Pseudomonas beneficially affected the development of plant-rhizobia symbiosis and its nitrogen-fixing activity, which though depended on the level of phosphorus nutrition (Table 4). In the case of insufficient phosphorus supply of the plants (0 and 5 mg P₂O₅/100 g substrate), P. fluorescens 21 didn’t affect the weight of plants and formation of root nodules, eventhough the significant increase in their nitrogen-fixing activity at introducing the first minimum dose of phosphorus fertilizer (5 mg P₂O₅/100 g substrate). The maximum nodulation and weight of plants were observed at the high dose  20 mg P₂O₅/100 g substrate. Inoculation of soybean with the mixed bacterial culture containing Pseudomonas against the phosphorus fertilizer resulted in a successive increase in green mass compared to that in the variant with pure culture of nodule bacteria. Probably, these conditions of phosphorus nutrition provided a positive effect owing to additional growth factors, such as phytohormones, manifested by Pseudomonas (12).
Thus, high yields of legumes crops inoculated at sowing with mixed bacterial cultures (nodule bacteria + bacteria the genus Pseudomonas) reflect the improved symbiotic nitrogen fixation and mineral nutrition in plants. The introduction of nitrogen and phosphorus fertilizers is necessary for obtaining the best results.

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