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, Rhizobiu lguinsarum bv. viceae 250, Bradyrhizobiu japonicum 110, R. meliloti 117, R. trifolii 348, and plant growth-promoting rhizobacteria of the genus Pseudomonas, . fluorescens 20 or . 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 117, 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 ( . % 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).

1. The yield of legumes inoculated at sowing with nodule bacteria and the bacteria of Pseudomonasgenera against the use of mineral fertilizers (microfield experiments; Moscow province, 1997-2006)

Variant (inoculum)

Fertilizer

Yield (g/vessel)

Grain
Peas, cv Smaragd

Rhizobium leguminosarum bv. viceae 250a

NPK

39,1

R. leguminosarum bv. viceae 250a + P. fluorescens 20

 

49,7

05

 

7,3

Soybean, cv Mageva

Not inoculated

NPK

18,7

Bradyrhizobiumjaponicu 110

NPK

49,1

B. japonicum 110 + P. fluorescens 20

NPK

58,0

B. japonicum 110 + P. fluorescens 21

NPK

59,5

05

 

5,3

Green mass (dry weight in total for 2 years)
Alfalfa, cv Slavyanskaya mestnaya

R. meliloti 117

NPK

171,9

R. meliloti 117 + P. fluorescens 20

NPK

227,9

05

 

35,0

Clover, cv VIK 1

Variant I

 

 

R. trifolii 348a

NPK

105,1

R. trifolii 348a + P. fluorescens 20

NPK

108,8

R. trifolii 348a + P. fluorescens 21

NPK

100,6

05

 

16,4

Variant II

 

 

R. trifolii 348a

PK

164,6

R. trifolii 348a + P. fluorescens 20

PK

191,2

05

 

25,9

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.

2. The mineral composition of nutrient elements transferred into yields of legumes inoculated at sowing with nodule rhizobia and the bacteria of Pseudomonas genera against the use of mineral fertilizers (microfield experiments; Moscow province, 1997-2006)

Variant (inoculum)

Fertilizer

N

P

K

Ca

Mg

Fe

Zn

Cu

Co

Mo

mg/vessel

ug/vessel

Soybean, cv Mageva (grain)

Not inoculated

NPK

895

127

187

45

64

1496

1646

898

116

112

Bradyrhizobiumjaponicu 110

NPK

3244

354

442

88

142

2946

3879

789

309

246

. japonicu 110 + .fluorescens 20

NPK

3486

389

505

104

168

3480

4814

1160

365

336

. japonicu 110+ . fluorescens 21

NPK

3683

434

518

107

173

3780

4841

1190

298

268

05

 

205

26

48

11

21

399

300

152

49

20

Alfalfa, cv Slavyanskaya mestnaya
(green mass in total for harvesting 3 times during the 1st growing season)

Rhizobiummeliloti 117

NPK

1420

189

1254

994

156

19600

7900

620

204

21

R. meliloti 117 +. fluoresces 20

NPK

2151

292

1682

1511

225

26100

11800

927

278

36

05

 

250

34

253

180

27

3700

1200

118

35

5

Clover, cv VIK 1
(green mass in total for harvesting 3 times during the 1st-2nd growing seasons)

R. trifolii 348

PK

3275

346

1866

2200

702

381

78

1238

2341

216

R. trifolii 348 +. fluorescens 20

PK

4109

394

2413

2588

802

440

101

1231

2795

263

05

 

686

45

289

320

94

53

13

199

365

41

3. The content of nitrogen (mg/vessel) fixed from the atmosphere, soil and fertilizers in legumes inoculated at sowing with nodule rhizobia and the bacteria of Pseudomonas genera against the use of mineral fertilizers (microfield experiments; Moscow province, 1997-2006)  

Variant (inoculum)

N from atmosphere

N from soil

N (15N) from fertilizer

Peas, cv Smaragd (grain)

Rhizobiumleguminosarum bv. viceae 250a

381

952

146

R. leguminosarumbv. viceae 250a + .fluorescens20

588

1126

166

05

95

119

16

Soybean, cv Mageva (grain + straw)

Not inoculated

845

156

Bradyrhizobiumjaponicum 110

2195

967

183

B. japonicum 110 + P. fluorescens 20

2473

1106

210

B. japonicum 110 + P. fluorescens 21

2399

1247

198

05

120

100

15

Note. A dash: in the variant without inoculation, the content of atmospheric nitrogen couldn’t be determined (calculated) owing to impossibility of comparison with non-inoculated control (11).

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%).

4. The yield of green mass, nodulation and nitrogen-fixing activity (NFA, ±) manifested by plant-rhizobia symbiosis in soybean (cv Mageva) at early fructification phase of the plants inoculated with Bradyrhizobiumjaponicum 110 + Pseudomonas fluorescens 21 against the different doses of phosphorus fertilizer (vegetation test).

Varian (inoculum)

Dose of P2O5,
mg/100 g
substrate

Green mass, g/vessel

Nodules

Number, pcs/vessel

Weight, g/vessel

NFA, mg 24/vessel š10-1

B. japonicum 110

0

11,6

45

3,1

0,4±0,3

B. japonicum 110 + P. fluorescens 21

0

10,6

38

3,1

0,2±0,1

B. japonicum 110

5

11,9

56

3,2

4,4±0,7

B. japonicum 110 + P. fluorescens21

5

12,6

56

3,6

44,9±12,3

B. japonicum 110

20

11,8

69

3,8

30,7±7,6

B. japonicum110 + P. fluorescens21

20

14,3

106

4,5

46,3±7,0

05

 

1,8

10

0,4

 

Note. a – the mean over 5 replications.

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.

REFERENCES

1. Umarov M.M., Assotsiativnaya azotficsatsiya (Associative Nitrogen Fixation), Moscow, 1986.
2. Belimov A.A. and Kozhemyakov A.P., Mixed Cultures of Nitrogen-Fixing Bacteria and Prospects for Their Use in Agriculture, S.-kh. biol., 1992, no. 5, pp. 77-87. 
3. Shabayev V.P., Smolin V.Yu. and Mudrik V.A, Nitrogen Fixation and CO2 Exchange in Soybeans (Glycine max L.) Inoculated with Mixed Cultures of Different Microorganisms, Biol. Fertil. Soils, 1996, vol. 23, no. 4, pp. 425-430.
4. Shavayev V.P., Productivity and Nitrogen Nutrition of Alfalfa Plants at the Combined Inoculation of Seeds with Rhizobium meliloti and Pseudomonas syringae pv. tabaci., S.-kh. biol., 2006, no. 3, pp. 67-73. 
5. Shabayev V.P. and Smolin V.Yu., Response of Winter Wheat to Inoculation with Pseudomonas Bacteria on Gray Forest Soil, Pochvovedenie, 2000, no. 4, pp. 497-504.
6. Shabayev V.P., Optimization of Doses of Mineral Fertilizers by Inoculation of Seeds with Growth Stimulating Rhizospheral Bacteria during Cultivation of Cereals, Dokl. RASKhN, 2004, no. 6, pp. 24-26.
7. Shabayev V.P., Safrina O.S. and Mudrik V.A., Influence of Rhizospheral Bacteria Pseudomonas florescens 20 and Endomycorrhizal Fungus Glous mosseae on Yield and Development of Radish Depending on Mineral Nutrition, Agrokhimiya, 1998, no. 6, pp. 34-41.
8. Shabayev V.P. and Smolin V.Yu., The Response of Fodder Beet to Inoculation with Pseudomonas fluorescens 20 in Gray Forest Soil as Conditioned by Mineral Nutrition Level, Pochvovedenie, 2002, no. 6, pp. 715-724.
9. Shabayev V.P., Response of Fodder Beet to Inoculation with Nitrogen-Fixing and Not-Fixing Pseudomonas on Alkaline Chernozem, S.-kh. biol., 2005, no. 3, pp. 55-59. 
10. Shabayev V.P. and Smolin V.Yu., Productivity of Rape and Transfer of Mineral Elements at Inoculation of Seeds with Pseudomonas fluorescens 20 Culture against Different Doses of Nitrogen Fertilizer, Agrokhimiya, 1999, no. 5, pp. 67-73.
11. Fosht D.D. and Poth M., Measurement of Biological Nitrogen Fixation by 15N Techniques, in Symbiotic Nitrogen Fixation Technology, Elkan G.N., Ed., Marcel Dekker Inc., New York - Basel, 1987, pp. 257-288.
12. Olyunina L.N. and Shabaev V.P., Producing of Indol-3-Acetic Acid during Growth of Rhizosphere Bacteria of the Genus Pseudomonas, Mikrobiologiya, 1996, no. 5, pp. 813-817.

 

The Institute of Physicochemical and Biological Problems of Soil Science, RAS,
Moscow province, Serpukhovsky district, Puschino 142290, Russia,
e-mail: VPSH@rambler.ru

Received June 3, 2008

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