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Creating a biological product using Nitrogen-fixing bacteria before sowing wheat (north Kazakhstan)

 

Zh.A. Baigonussova1*, S.A. Tulkubaeva2, Yu.V. Tulaev2, O.S. Safronova2, A.A. Kurmanbaev1

 

1RSE on REM "National Center for Biotechnology" KN MES, Nur-Sultan, Republic of Kazakhstan. 2Zarechnoye Agricultural Experimental Station LLP Republic of Kazakhstan, Kostanay region, Zarechnoye, Republic of Kazakhstan.

 

Correspondence: Zh.A. Baigonussova, RSE on REM "National Center for Biotechnology" KN MES, Nur-Sultan, Republic of Kazakhstan.


ABSTRACT

Based on the associations of nitrogen-fixing bacteria of the Rhizobium sp. and Azotobacter sp. genera, biofertilizers have been developed for increasing the wheat yield in Northern Kazakhstan. Among nodule bacteria and azotobacters, nine promising strains of microorganisms that can stimulate plant growth, fix nitrogen, and exhibit antagonism to phytopathogenic micromycetes have been isolated. Three associations have been tested in the vegetation experiments. According to the results of the experiment, association No. 2 that was tested in field studies upon introduction before sowing spring wheat was selected. The biofertilizer has increased the wheat grain yield by 275 kg/ha.

Keywords: Biofertilizer, Nitrogen-fixing bacteria, Nodule bacteria, Biocompatibility, Antifungal activity, Field experiment


Introduction

 

Kazakhstan has a significant potential for developing organic production due to the low level of using mineral fertilizers and pesticides and the availability of significant areas of fertile chernozem soil in the north of the country. The agro-industrial complex of Kazakhstan can become one of the world's largest producers of agricultural export products, especially organic food. All necessary political, legal, and scientific prerequisites for organic farming development are in place. For instance, within the framework of the Concept for the transition to a green economy, the law "On the production of organic products" has been adopted, the Organic Center is operating under the aegis of the Ministry of Agriculture of the Republic of Kazakhstan, technologies of biological farming are being developed, and the interest of agricultural companies to organic production is growing due to the higher price of organic products. The demand for ecologically clean food is growing 1.5 – 2 times faster than the supply, which gives Kazakhstan great opportunities due to the availability of clean land [1]. 

Organic farming is based on reducing or completely weaning off the use of synthetic mineral fertilizers, plant protection chemicals, plant growth regulators, and genetically modified organisms, and the maximum use of biological products and biotechnologies at all stages of agricultural production [2].

Complex biological products that combine the properties and functions of various biological products are preferable [3].

Practice shows that the use of complex biofertilizers contributes to a stable yield growth by 25 – 50% and more with significantly reduced root rot of the plants [4, 5]. The fungistatic effect is usually there because the level of soil biodiversity grows significantly, while the importance of unwanted populations (for example, phytopathogenic fungi) decreases.

The meta-analysis of the data about the biological products intended for agriculture showed the advantage and effectiveness of biofertilizers based on nitrogen-fixing and phosphate-mobilizing bacteria [6].

In the scientific literature, there are several reports about attempts to create biofertilizers based on associations of nitrogen-fixing bacteria of the Rhizobium and Azotobacter genera.

For instance, Korniichuk et al. claimed to develop a promising biofilm biofertilizer from the free-living bacteria of the Rhizobium and Azotobacter genera, which positively influenced the mass of lettuce sprouts, providing a 49.6% increase in the biomass, compared to untreated reference lettuce. The positive effect was due to the nature of the bacteria of the PGPR group [7].

An important property of the Azotobacter genus is the ability of some species to degrade environmental pollutants. It should be noted that the Azotobacter bacteria combine well with the bacteria of the Clostridium, Pseudomonas, Bacillus, Azospirillum, Agrobacterium genera, and, most importantly, rhizosphere bacteria (the Rhizobiaceae family) [8].

For Kazakhstan, the most important crop is spring wheat. The concept of developing a green economy in the Republic of Kazakhstan sets the task of significantly increasing the yield of this crop. Biofertilizers and biopesticides can play an important role in accomplishing this task [9].

In this work, the goal was developing a nitrogen-fixing bacteria-based biofertilizer for wheat that would be suitable for Northern Kazakhstan.

 

Materials and Methods

 

Soil samples were taken using the envelope method from the rhizosphere of plants in the crops of wheat, corn, lentils, and peas of LLP Agricultural Experimental Station Zarechnoye, and the Dostyk peasant farm. The pure cultures of the bacteria of the Azotobacter genus were isolated from the soil using the method of lumps according to Vinogradsky [10]. Nodule bacteria were isolated from the lentil nodules selected in the flowering phase from large and dense nodules.

The objects of the study were Rhizobium leguminosarum nitrogen-fixing nodule bacteria, Rh-1, Rh-2, Rh-5 strains, and bacteria of the Azotobacter sp. genus, Az-34, Az-4, Az-6, Az-20, Az-28, Az-13, Az-1/2 strains, obtained by the authors earlier from the rhizosphere of agricultural plants. Nitrogen-fixing bacteria were grown on Burk's medium, while nodule bacteria — in the agar medium with bean broth.

Nitrogen fixation of the bacteria was determined by the accumulation of ammonium in the medium [11]. The nitrogen-fixing activity of the isolated strains was determined based on measuring the concentration of free NH4+ ions in the culture liquid.

The biofertilizer was tested upon sowing spring wheat of the Omskaya-36 variety.

Chemical fungicide Lamador manufactured by Bayer Schering Pharma, USA was used for seed treatment.

The antifungal activity of the bacteria to Fusarium sp., Alternaria alternata was determined using the method of wells in the joint cultivation of the bacteria and phytopathogens on the Sabouraud and Chapek-3 media [12].

The strains were identified by determining the direct nucleotide sequence of the 16S rRNA gene fragment, followed by comparing the nucleotide identity with the sequences deposited in the international GeneBank database [13].

To study the effect of liquid preparation forms of bacterial fertilizers on the growth of grain crops, a vegetation experiment was laid in a greenhouse of the National Center for Biotechnology in Nur-Sultan.

Field experiments were laid on a fallow field, the soil was southern chernozem. The field was prepared and the experiments were laid as per the relevant recommendations [14], with separate additions and changes adopted at LLP Agricultural Experimental Station Zarechnoye, the soil in the fallow field was southern chernozem.

Observation and accounting, land plot selection and preparation, laying and performing the experiment, and statistical processing of the results were performed according to Dospechov  [15].

The percentage of protein, gluten, and moisture was measured using an IR analyzer at an ambient temperature of 21 °C (18 25) and relative humidity of 64% (30-80%) [16]. The quantitative protein content was determined following the state industry standard. For the mathematical processing of the results, standard methods for finding mean values and their mean errors were used [17].

 

Results and Discussion

 

Studying the cultural properties of the strains of nitrogen-fixing bacteria

The morphological and biochemical properties of nine selected strains of azotobacter and nodule bacteria were studied. Following the cultural and phenotypic properties, six strains (Az-4/3, Az13, Az-6, Az-2/1, Az-9/2, and Az-34) were assigned to the Azotobacter genus according to Bergey's Manual. The strains produced a water-insoluble brown pigment, had peritrichous flagella and formed cysts. On Ashby's medium, the strains formed large, round, convex, and slimy colonies of 7 to 13 mm in diameter with a brown pigment and smooth edges. With aging, the colonies became slimmer and spread over the agar surface.

The strains of nodule bacteria (Rh-1, Rh-2, and Rh-5) formed convex, translucent, and slimy, rounded with smooth edges colonies with a diameter of 4 – 7 mm of a light-beige color. The cells of nodule bacteria were gram-negative, 0.5 – 1.2 µm in diameter, motile, rod-shaped, and aerobic.

Biochemical tests on nine strains showed the absence of amylase activity in bacterial strains; gelatin was liquefied by Az-13 and Rh-5 strains. The catalase activity was observed in all strains; high activity was observed in Az-34, Az-3, and Az-6 strains. The oxidase activity was observed in Az-34, Az-6, and Az-13 strains. When growing microorganisms on a medium with ferrous ammonium citrate, the formation of iron sulfide (FeS) was found in one strain (Rh-1). None of the studied strains produced ammonia. The studied strains assimilated carbohydrates and alcohols. The optimal conditions for growing strains are the following: the temperature within 25 – 28 °C and the medium acidity within pH 7.0 – 7.4 (Table 1).

 

 

Table 1. Physiological and biochemical properties of the strains of azotobacter and nodule bacteria

The name of the strains

Relative to oxygen

Test catalase

Test an oxidase

Gelatin Liquefaction
Hydrolysis of starch
Hydrogen sulfide formation
Indole formation
Ammonia formation

Growth on the medium Ashby

Utilization

sucrose

lactose

galactose

mannose

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Az-34

A

+

+

-

-

-

-

-

+

-

+ O2

-

-

Az-6

A

+

+

-

-

-

-

-

+

+ O2

-

+ O2

+ O2

Az-4/3

A

+

+

-

-

-

-

-

+

-

+ O2

-

+

Az-9/2

A

+

-

-

-

-

-

-

+

±

+ O2

±

±

Az-2/1

A

+

+

-

-

-

-

-

+

-

+ O2

-

+

Az-13

A

+

+

-

-

-

-

-

+

+ O2

+ O2

+ O2

+ O2

Rh-1

A

+

+

-

-

-

-

-

+

+

±

±

±

Rh-5

A

+

+

+

-

+

-

-

+

+

±

±

±

Rh-2

A

+

-

+

-

+

-

-

+

+

±

±

±

Notice: “A” aerobe; “+” positive reaction; “-” negative reaction; “ ± " weakly expressed by the positive response; "+ O2” positive response forming oxygen.

 

 

Genetic identification of the studied bacterial strains based on the analysis of the nucleotide sequence of the 16S rRNA gene

The nucleotide sequences of the 16S rRNA gene of the identified strain were analyzed and combined into a common sequence in the SeqMan software (Applied Biosystems). After that, the terminal fragments (nucleotide sequences of primers, fragments with low-quality indices) were removed, which allowed obtaining a nucleotide sequence with a length of more than 650 bps, which was identified in GeneBank using the BLAST algorithm. The results of the genetic identification of the studied strains are shown in Table 2.

 

Table 2. The results of identification using the method of analyzing the nucleotide sequence of the 16S rRNA gene using BLAST http://blast.ncbi.nlm.nih.gov/

No.

Strain names

Generic affiliation

% of identity

1

Az-13

Azotobacter chroococcum

99.80%

2

Rh-1

Rhizobium radiobacter

99.40%

3

Az-4/3

Azotobacter chroococcum

100.0%

4

Az-6

Beijerinckia fluminensis

100.00%

5

Az-9/2

Azotobacter chroococcum

97.10%

6

Az-2/1

Achromobacter marplatensis

100.00%

7

Az-34

Agrobacterium tumefaciens

98.53%

8

Rh-2

Rhizobium legmunasarium.

98.10%

9

Rh-5

Agrobacterium sp.

99.29%

 

The evolutionary relationships of taxa

The evolutionary history was derived using the Neighbor-Joining method [18]. An optimal tree with the sum of the branch length = 0.06861339 was shown (next to branches). The evolutionary distances were calculated using the maximum composite likelihood method [19] and expressed in terms of the number of base substitutions on site. This analysis included 13 nucleotide sequences. All ambiguous positions were removed for each pair of sequences (the pairwise deletion option). There was a total of 1,483 positions in the final dataset. Evolutionary analysis was performed in MEGA X [20] (Figure 1).

 

 

 

Culture 2_page-0001

Culture 4_page-0001

Figure 1. The phylogenetic tree of isolated bacterial strains from the rhizosphere of wheat and pea nodules

 

 

Studying the biological activity of the strains of nitrogen-fixing bacteria

The assay of the nitrogen-fixing ability of the bacteria was performed using the method of screening the studied strains grown on a nitrogen-free mineral medium (NFMM) using glucose as a carbon source (Figure 2).

 

 

Figure 2. The assay of the nitrogen-fixing ability of bacterial strains on NFMM

 

 

The nitrogen-fixing activity was visually detected using NFMM with 0.7% glucose. Bromothymol blue (BTB) was added to the medium as an indicator. After seven days of incubation, a medium color change was noted.

The nitrogen-fixing ability of the bacterial strains was established by the yellow-green coloration of the medium, which showed the pH changes in the alkaline range; these pH changes were associated with the accumulation of ammonia ions [21].

The nitrogen-fixing ability of the bacterial strains was measured based on the measurement of the concentration of NH+4 free ammonium ions, which showed that the amount of fixed nitrogen in the culture medium varied from 4.5 to 41.027 μmol/l of NH+4 (Table 3).

 

Table 3. Measuring ammonia accumulation by Azotobacter sp. and Rhizobium sp. strains

No.

Strain name

Empty test tube weight

Gross wet weight

Net wet weight

OD540

conc. of NH4+ μmol/l

1

2

3

4

5

6

7

1

Rh-1

0.995

1.1787

0.1787

0.569

41.0

2

Rh-4

0.991

1.1043

0.1133

0.831

14.9

3

Az-34

0.996

1.0515

0.0603

0.284

5.1

4

Az-4/3

0.987

1.0415

0.0545

0.273

4.9

5

Az-6

0.991

1.029

0.0380

0.454

8.2

6

Rh-5

0.989

1.1139

0.1249

0.086

1.6

7

Az-9/2

0.946

1.0321

0.0861

0.058

1.0

8

Az-13

0.933

1.0291

0.0889

0.064

1.0

9

Az-2/1

0.946

1.0232

0.0772

0.023

4.5

 

By the ability to accumulate nitrogen, the most active were Rhizobium leguminosarum Rh-1, Rh-4, and Azotobacter chroococcum Az-34, and Az-4/3.

 

Studying the antifungal activity of the strains of nitrogen-fixing bacteria

A study of the antifungal activity of azotobacter and nodule bacteria cultures showed that all strains suppressed and inhibited the growth of fungal mycelium. This allows using these strains in fighting the pathogens of crops (Figure 3).

 

 

Figure 3. The zones of the A. alternate test fungus growth inhibition with the tested cultures (Czapek-3 agar medium (perforation method))

 

 

Studying the strains' ability to stimulate the growth of plants

In the laboratory and vegetation experiments where radish seeds were treated with Az-12, Az-8/9, Az-29, Az-24, Az-23, Az-6, Rh-2, Rh-3, Az-36, Az-28, Az-13, Az-25, and Az-22 bacterial strains, an increase in seed germination rate up to 85 – 100% was observed, while in the reference, the germination rate was only 35%. 100% germination and stimulation of root and seedling growth were ensured by Az-4, Az-28, Rh-1, Az-6, and Rh-3 bacterial strains (Figure 4).