CHARACTERIZATION OF BRADYRHIZOBIA ASSOCIATED WITH SOYBEAN PLANTS GROWN IN UKRAINE

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April – May 2020, vol. 9, no. 5
pages: 983-987
Article type: Microbiology of Microbiology
DOI: 10.15414/jmbfs.2020.9.5.983-987
Abstract: The phenotypic and genotypic features of soybean nodule bacteria with different growth rates common in soils of Ukraine have been studied. The obtained results suggest high heterogeneity of soybean microsymbionts. It has been established that all studied strains are able to form active symbiotic relationships with soybean. When interacting with Chinese cowpea, intensive-growing strains form an ineffective symbiosis with a low nitrogen fixation level (phenotype Nod+Fix–), while slow-growing strains form complete nitrogen fixing systems (phenotype Nod+Fix+). Upon cultivation in the mannitol yeast medium, intensive-growing strain B. japonicum KC19 produces a significant amount of auxins and cytokinins, while the slow-growing strain B. japonicum KC23 is significantly superior by the amount of gibberellins. According to the organization of the genome defined in REP-PCR, studied strains of soybean rhizobia belong to two genetic groups, which corresponds to the division of strains into groups by the growth rate (slow- and intensive-growing). Based on the RFLP analysis of rpoB gene, using restriction enzymes MspI, HaeIII and NdeII, intensive-growing strains were combined into one rpoB type, while slow-growing strains belong to 2-3 rpoB types.
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CHARACTERIZATION OF BRADYRHIZOBIA ASSOCIATED WITH SOYBEAN PLANTS GROWN IN UKRAINE


AUTHORS

Dmytro Krutylo, Natalia Leonova, Olena Nadkernychna

ABSTRACT

The phenotypic and genotypic features of soybean nodule bacteria with different growth rates common in soils of Ukraine have been studied. The obtained results suggest high heterogeneity of soybean microsymbionts. It has been established that all studied strains are able to form active symbiotic relationships with soybean. When interacting with Chinese cowpea, intensive-growing strains form an ineffective symbiosis with a low nitrogen fixation level (phenotype Nod+Fix–), while slow-growing strains form complete nitrogen fixing systems (phenotype Nod+Fix+). Upon cultivation in the mannitol yeast medium, intensive-growing strain B. japonicum KC19 produces a significant amount of auxins and cytokinins, while the slow-growing strain B. japonicum KC23 is significantly superior by the amount of gibberellins. According to the organization of the genome defined in REP-PCR, studied strains of soybean rhizobia belong to two genetic groups, which corresponds to the division of strains into groups by the growth rate (slow- and intensive-growing). Based on the RFLP analysis of rpoB gene, using restriction enzymes MspI, HaeIII and NdeII, intensive-growing strains were combined into one rpoB type, while slow-growing strains belong to 2-3 rpoB types.


KEYWORDS

Bradyrhizobium japonicum, phytohormones, HPLC, REP-PCR, RFLP analysis, rpoB gene, soybean

INTRODUCTION

Nodule bacteria (rhizobia) are the large group of microorganisms that are able to form a symbiotic relationship with leguminous plants, forming root nodules.

Recently, increasing attention has been paid to the study of soil populations of microsymbionts of different legumes (de Bruijn, 2015; Chidebe et al., 2017). Under regular circulation between ecological niches (soil → nodules → soil) in populations of nodule bacteria, significant transformations of their spatial and genetic structures develop. Such changes can lead to the emergence of new genotypes of rhizobia (Provorov, 2009). To understand the mechanisms responsible for the formation of the genetic diversity of nodule bacteria in natural ecosystems and agrocenoses, detailed study of the representatives of the soil populations of these microorganisms is required.

The most common leguminous culture of world agriculture is soybean (Glycine max (L.) Merr.). Nodule bacteria of different genera: Bradyrhizobium, Sinorhizobium (Ensifer) and Mesorhizobium may to form a symbiotic relationship with the soybean plant. They are represented by species of bacteria with different growth rates: slow-growing B. japonicum (Jordan, 1982), B. elkanii (Kuykendall et al., 1992), B. liaoningense (Xu et al., 1995) and B. yuanmingense (Yao et al., 2002) and fast-growing Ensifer (Sinorhizobium) fredii, S. xinjiangensis (Peng et al., 2002) and M. tianshanense (Chen et al., 1995).

It should be noted that the formation of local populations of soybean nodule bacteria in Ukrainian soils began relatively recently – since the intense introduction of soybeans in crop rotation and the use of biopreparations on the basis of specific microorganisms. Our studies have shown that soil soybean rhizobia populations differ in density and qualitative composition, and they are quite heterogeneous. As a result of the analysis of morphological, cultural, physiological, biochemical and chemotaxonomic properties of 180 strains of soybean rhizobia, isolated from soils of different regions of Ukraine, we have identified nodule bacteria, which according to genetic features belong to the species B. japonicum, but differ significantly from the typical slow-growing soybean microsymbionts (Patyka et al., 2010; Krutylo and Zotov, 2015). Isolated strains are characterized by increased growth rates and conventionally called “intensive-growing strains”. At day 5-6 after sowing, intensive-growing strains form large (2-4 mm diameter) translucent colonies with matte shading on the legume medium. Representatives of the second group of strains (slow-growing) accumulate bacterial mass at day 8-10 days and form small (up to 1 mm diameter) white colonies.

It has been established that intensive-growing soybean nodule bacteria are characterized by increased survival rate in the soil, but do not occur in all regions of Ukraine. The ratio between the representatives of the two groups of strains varies depending on the soil-climatic zone and the duration of soybean cultivation (Patyka et al., 2010).

Considering the above, the objective of our work was to study the phenotypic and genotypic features of soybean nodule bacteria in soils of Ukraine.

MATERIAL AND METHODS

Microorganisms

180 strains of soybean nodule bacteria were isolated from different soil types of Ukraine (Regions of Chernihіv, Vinnytsia and Sumy) by sterile germ inoculation method. In the result of comparative analysis of morphological, cultural, physiological and biochemical properties, these strains were divided into two groups: intensive- and slow-growing strains. The objects of this study were strains – typical representatives of soybean nodule bacteria with slow (B. japonicum 46, KC23, KH10) and intensive (B. japonicum KB11, KC19, KC22) growth rates. The typical strain of slow-growing nodule bacteria B. japonicum VKM B-1967 = USDA 6T = ATCC 10324Т also used in this work. Rhizobial strains are stored in the collection of the Laboratory of Plant-Microbial Interactions and in the Collection of useful soil microorganisms at the Institute of Agricultural Microbiology and Agroindustrial Manufacture of the National Academy of Sciences of Ukraine (B. japonicum 46 = B-23, KH10 = B-124).

Vegetation experiments conditions

In vegetation experiments, we studied the ability of soybean nodule bacteria to form a symbiotic relationship with soybean (Glycine max (L.) Merr.) and cowpea (Vigna unguiculata (L.) Walp.). Cultivation of nodule bacteria was carried out in flasks (750 ml) on liquid medium (for 72 hours), containing (g/l): К2НРО4 – 0.5, КН2РО4 – 0.5, (NH4)2SO4·7H2O – 1.0, MgSO4·7H2O – 0.2, NaCl – 0.2, CаCO3 (sterile) – 0.1, sucrose – 2.0, mannitol – 3.0, glucose – 10.0, broth of peas (peas seeds – 50 g per 1 litre of water) – 100.0 ml/l; pH 7.0-7.2. The titre of bacteria was 2·109 CFU/ml. Inoculation load was 200–300 thousand cells per 1 seed. Experiments were conducted in the greenhouse in 2.5 L vessels. The sterile vermiculite was used as a nitrogen-free substrate. The repetition of the experiment was sixfold. Activity of symbiotic nitrogen fixation was measured in flowering phase by acetylene method (Hardy et al., 1968).

Assay of phytohormones activity

Cultivation of bacteria was carried out in flasks (750 ml) on a rotary shaker (220 rev/min) at 26-28˚C on liquid mannitol–yeast digest medium with such composition (g/l): mannitol – 10.0; yeast extract – 2.0; calcium gluconate – 1.5; K2HPO4 – 0.5; MgSO4 – 7H2O – 0.2; NaCl – 0.1; FeCl3 6H2O – 0.01; pH 7.2. Cultures of B. japonicum in the exponential growth phase were used as the inoculum. The amount of inoculum was 5% volume of the medium.

To separate the biomass, the cultural liquid of bacteria was centrifuged for 20 min at 9000 rpm and at + 4˚C. Cells of bacteria were washed with saline solution thrice in order to clean them from exopolymer residues, each time they were centrifuged under the same conditions. The supernatants were used for further research to extract phytohormonal compounds. The cells biomass was suspended in distilled water, then dried at 103-105˚C in a desiccator until constant weight. The amount of absolutely dry biomass (ADB) of microorganisms was determined gravimetrically.

Extracellular phytohormones were isolated from supernatants of nodule bacteria by the method described in (Methodical recommendations by phytohormones definition, 1988). Assay of phytohormones in the cultural medium was performed using high performance liquid chromatography (HPLC) on liquid chromatograph Agilent 1200 LC (USA) and mass spectral detector Agilent G1956B.

The standards were synthetic phytohormones SigmaAldrich (Germany) and Acros Organic (Belgium): auxins – indole-3-acetic acid, indole-3-butyric acid, indole-3-carboxylic acid, indole-3-carbinol, indole-3-acetic hydrazide, indole-3-carboxaldehyde; cytokinins – zeatin, trans-zeatin-riboside, kinetin, (N6-(2-Isopentenyl)adenine, N6-(2-Isopentenyl)adenosine; abscisic acid; gibberellins – GA3, GA4.

Chromatogram calculation was performed using the ChemStation software. Amount of phytohormones was calculated in µg per 1 g of absolutely dry biomass of producer.

DNA isolation

Soybean nodule bacteria were cultivated in agar medium TY (Beringer, 1974). The total DNA of the rhizobia strains was isolated from the fresh cultures (the exponential growth phase) by the lysis of the bacterial cells lysozyme-SDS with further phenol-chloroform extraction and precipitation with the use of isopropanol (Laguerre et al., 1992).

REP-PCR analysis

REP-PCR (repetitive extragenic palindromic sequences) was performed for genotyping and to detect the identity of soybean nodule bacteria strains. The amplification was conducted with the use of REP-primers: REP1R-I (5′-iiiicgicgicatciggc-3′) and REP2-I (5′-icgicttatciggcctac-3′). PCR were performed using the standard reaction mixture (Versalovic et al., 1994). The temperature-time profile of amplification: denaturation at 94°C for 3 min, 40 cycles consisting of 30 sec at 94°C, 30 sec at 37°C, 3 min at 68°C, final elongation for 5 min at 68°C. The PCR products were separated by horizontal electrophoresis on 1.5% agarose. Gels were stained with ethidium bromide, visualized under UV radiation and photographed.

RFLP analysis of the rpoB gene

The amplification of the rpoB gene of soybean nodule bacteria was conducted with the use of primers rpoB83F: 5′-cctcatcgaggttcagaaggc-3′ and rpoB1061R: 5′-agcgtgttgcggatataggcg-3′ (Martens et al., 2008). The temperature-time profile of amplification: denaturation at 94°C for 5 min, 4 cycles consisting of 2 min at 94°C, 2 min at 58°C, 1 min at 72°C, and them 31 cycles consisting of 30 sec at 94°C, 1 min at 58°C, 1 min at 72°C, final elongation for 7 min at 72°C.

The restriction analysis (RFLP) was carried out with the use of restriction endonuclease MspI, HaeIII, NdeII (Fermentas, USA) according to the manufacturer’s instruction. DNA processed by restrictase was analyzed with the use of electrophoresis in 2.5% agarose gel.

Statistical data analysis

Microsoft Excel was used to analyze data on the average of four replicates (±SE).

Comparison of REPPCR fingerprints was performed using FPQuest software (Bio Rad, USA). The degree of similarity of the fingerprints was assessed by Pearson’s correlation coefficient.

Fingerprint analysis of the RFLP profiles was carried out using Total Lab v. 2.01 software. Following installation of the gel images into the program, the bands were determined with their pixel positions and their molecular weights were scored according to the molecular size marker.

RESULTS AND DISCUSSION

Symbiotic characteristic of soybean nodule bacteria

In the work, strains of soybean nodule bacteria with slow and intense growth, common in soils of different regions of Ukraine were used (Tab 1). Under vegetative experiments, the specificity of the symbiosis of slow- and intensive-growing B. japonicum strains with two legume cultures was studied: soybean as a host plant, and Chinese cowpea. It is known that cowpea belongs to a symbiotically unspecialized plant species and can form nodules with representatives of several genera of nodule bacteria: Rhizobium, Bradyrhizobium, Mesorhizobium (Fotev et al., 2016; Tampakaki et al., 2017). Therefore, studied strains of soybean rhizobia may be potential microsymbionts of this plant.

The analysis of the results showed that all strains, regardless of the growth rate, formed effective symbiosis with soybean (phenotype Nod+Fix+), forming nodules with high nitrogen fixation activity (25.9-30.5 μg N per plant per 1 hour). We have detected significant differences between strains when they interact with Chinese cowpea. Slow-growing B. japonicum strains VKM B-1967, 46, KH10, KC23 formed well-developed nitrogen fixing nodules (phenotype Nod+Fix+) on the roots of plants, whereas intensive-growing strains of B. japonicum KB11, KC19, and KC22 predominantly formed inactive white nodules (phenotype Nod+Fix). Nitrogenase activity of the cowpea nodules in the variants with inoculation using intensive-growing B. japonicum strains was 4.3-6.1 times lower compared to the slow-growing strains with Nod+Fix+ phenotype, which fixed the molecular nitrogen at 13.0-21.9 μg N per plant per 1 hour. We believe that revealed differences may indicate the formation of a nonspecific symbiosis between intensive-growing strains of soybean rhizobia and Chinese cowpea.

Table 1 Symbiotic characteristic of soybean nodule bacteria with different growth rate intensity

Strains Geographical origin Symbiotic phenotypes
Glycine max Vigna unguiculata
B. japonicum VKM B-1967 * Japan Nod+Fix+ Nod+Fix+
B. japonicum 46* Ukraine, Vinnytsia region Nod+Fix+ Nod+Fix+
B. japonicum КH10* Ukraine, Chernihіv region Nod+Fix+ Nod+Fix+
B. japonicum КC23* Ukraine, Sumy region Nod+Fix+ Nod+Fix+
B. japonicum КB11 Ukraine, Vinnytsia region Nod+Fix+ Nod+Fix
B. japonicum КC19 Ukraine, Sumy region Nod+Fix+ Nod+Fix
B. japonicum КC22 Ukraine, Sumy region Nod+Fix+ Nod+Fix

Legend: * – slow-growing strains of soybean nodule bacteria, Nod+Fix+ – formation of nitrogen-fixing nodules; Nod+Fix – formation of non-nitrogen fixing nodules.

Thus, studied soybean nodule bacteria with slow and intense growth differ in their symbiotic specificity.

Phytohormones activity

It is known that phytohormonal substances have a significant influence on the symbiosis of microorganisms and macroorganisms. Study of the spectrum phytohormones synthesised by the strains of nodule bacteria is important in evaluating their symbiotic potential and identifying the features of interaction with host plants (Spaepen et al., 2007; Leonova et al. 2013; Held et al., 2014). In our work, we studied the ability of soybean rhizobia strains with slow (B. japonicum KC23) and intense (B. japonicum KC19) growth to synthesise biologically active substances.

HPLC analysis showed that during cultivation in the mannitol yeast medium, B. japonicum strains synthesised phytohormones of auxin, cytokinin and gibberellic nature (Tab 2).

Table 2 Production of extracellular phytohormones by Bradyrhizobium japonicum strains with different growth rate

Phytohormones Amount of phytohormones, µg/g of absolutely dry biomass
slow-growing strain

B. japonicum КС23

intensive-growing strain

B. japonicum КС19

Auxins, including: 1.6 44.7
indole-3-acetic acid (IAA) 0.1 5.0
indole-3carbinol (IC) 4.3
indole-3-butyric acid (IBut)
indole-3-acetic hydrazide (IAA-hydr.) 28.3
indole-3-carboxaldehyde(ICal) 0.8
indole-3-carboxylic acid (ICA) 0.7 7.1
Cytokinins, including: 1.4 17.0
kinetin (Kin) 12.5
zeatin (Z) 0.1 1.3
trans-zeatin-riboside (ZR)
N6-(2-Isopentenyl)adenine (IPA)
N6-(2-Isopentenyl)adenosine (IPAr) 1.3 3.2
Gibberellins, including: 205.5 85.0
GA3 201.2 85.0
GA4 4.3
Abscisic acid

Legend: «–» phytohormones not detected.

The quantitative and qualitative differences between strains on the level of synthesis stimulating action phytohormones have been detected. Intensive-growing strain B. japonicum KC19 synthesised significantly more auxins (44.7 µg/g ADB) and cytokinins (17.0 µg/g ADB) compared to the slowly-growing strain B. japonicum KC23 (1.6 and 1.4 µg/g ADB, respectively). A supernatant of the intensive-growing strain presented a wider range of phytohormonal substances of auxin and cytokinin nature. However, the slow-growing strain was significantly superior by gibberellin synthesis (205.5 µg/g ADB) compared to intensive-growing strain of B. japonicum KC19 (85.0 µg/g ADB).

Both strains B. japonicum KC23 and B. japonicum KC19 did not produce abscisic acid, which is a phytohormone able to inhibit plant growth and development. It is known that some representatives of B. japonicum species are capable of producing abscisic acid (Kots et al., 2010).

Therefore, the studied strains of soybean nodule bacteria differ in the level of the synthesis of extracellular phytohormones: intensive-growing strain B. japonicum KC19 produces a higher amount of auxins and cytokinins, whereas the slow-growing strain B. japonicum KC23 is significantly superior by the amount of gibberellins. The revealed differences can play an important role in the processes of root colonization by the rhizobia and formation of nodules.

REPPCR amplification

It is known that REP elements (short repetitions of 32 bp) are widely used as targets for the identification of microorganisms. The result of the amplification reaction of the bacterial genome sites between the REP elements is the formation of a set of DNA fragments separated in agarose gel, which reflect the organization of the genome of a particular microorganism (Judd et al., 1993; Kozyrovskaya and Kovtunovich, 1994).

For determination of genetic heterogeneity and detection of related groups in strains of soybean nodule bacteria, their genotyping was performed using REP-PCR. The genomic fingerprints of seven B. japonicum strains with slow and intense growth, obtained by amplification of their DNA with RER primers, were analysed (Fig 1).

Figure 1 REP-PCR patterns of B. japonicum strains. Lane M – 100 bp molecular weight marker (GeneRuler 100bp DNA Ladder, Fermentas); lanes 1, 2, 3 – strains with intensive growth rate (КВ11, КС19, КС22); lane 4 – type strain B. japonicum VKM B-1967; lanes 5, 6, 7 – strains with slow growth rate (46, КС23, КН10).

Cluster analysis of REP-PCR profiles allowed to detect significant differences in the genetic organization of soybean nodule bacterial strains with different growth rates. For example, by the DNA profiles, the studied strains were reliably divided into two clusters (Fig 2). Genetic group I included intensive-growing strains similar by RER profiles. Genome group II includes all the slow-growing strains, including the typical strain of B. japonicum VKM B-1967.

 

Figure 2 Dendrogram showing genetic relationships among B. japonicum strains with different growth rate using REP profiles: 1, 2, 3 – strains with intensive growth rate (KB11, KC19, KC22); 4 – type strain B. japonicum VKM B-1967; 5, 6, 7 – strains with slow growth rate (46, KC23, KH10).

Within the identified genetic groups, we have also noted the variability of the nodule bacteria. For example, intensive-growing strain of B. japonicum KB11 differed from intensive-growing strains of B. japonicum KC19 and B. japonicum KC22, which had identical fingerprint patterns. It has been proved that the number of DNA fragments studied slow-growing strains are genetically different from the typical strain of B. japonicum VKM B-1967. The same REP profiles were typical for slow-growing strains of B. japonicum 46 and B. japonicum KH10.

Therefore, according to the results of genotyping, the studied strains of soybean rhizobia are genetically heterogeneous. Two separate groups of related nodule bacteria were identified, which corresponds to the division of strains into groups by the growth rate (slow and intense growth).

PCRRFLP analysis of the rpoB gene

Recently, in addition to 16S rRNA and 16S-23S rRNA intergenic spacer region (ITS), the analysis of functionally important genes (housekeeping genes) is widely used to identify microorganisms and assess the diversity. One of these genes is the rpoB gene encoding RNA polymerase β-subunit and used as a highly conserved marker (Martens et al., 2008; Rivas et al., 2009).

We have studied a restriction fragments length polymorphism of the rpoB gene in five strains of soybean nodule bacteria with different growth rates. By amplification of the gene in all strains, one fragment approximately 900 bp long was formed, which was then separately cleaved with restriction enzymes MspI, HaeIII and NdeII.

The restriction analysis of rpoB gene amplification products allowed to reveal a high degree of heterogeneity of studied soybean rhizobia, and as the result they were classified as different rpoB types (Tab 3).

Table 3 RFLP types and restriction fragments determined by PCR-RFLP analysis of rpoB gene of B. japonicum strains

Strains Restricted fragments size (pb) rpoB types
MspI
B. japonicum KB11 140–170–260 MI
B. japonicum KC19 140–170–260 MI
B. japonicum VKM B-1967* 140–160–170–260 MII
B. japonicum 46* 140–170–260 MI
B. japonicum KC23* 140–170–260 MI
HaeIII
B. japonicum KB11 65–90–130–160–370 HI
B. japonicum KC19 65–90–130–160–370 HI
B. japonicum VKM B-1967* 65–90–100–160–525 HII
B. japonicum 46* 65–90–130–160–525 HIII
B. japonicum KC23* 80–160–200–525 HIV
NdeII
B. japonicum KB11 120–170–500 NI
B. japonicum KC19 120–170–500 NI
B. japonicum VKM B-1967* 120–170–200–300 NII
B. japonicum 46* 120–170–200–300 NII
B. japonicum KC23* 100–120–130–170–200–300 NIII

Legend: * – slow-growing strain of soybean nodule bacteria

Upon the use of the MspI enzyme, intensive-growing strains (B. japonicum KB11, KC19) and slow-growing strains (B. japonicum 46, KC23) had the same restriction profiles and formed one rpoB type (MI). A typical strain of B. japonicum VKM B-1967 differed from the rest of the strains, and was classified as MII rpoB type.

Significant differences between strains were detected under the use of restriction endonucleases HaeIII and NdeII.

Following cleavage of rpoB gene by HaeIII restriction enzymes, four to five fragments of different sizes were formed in the studied rhizobia, which allowed the strains to be combined into four restriction types – HI-HIV. One fragment of 160 bp in size was found in all nodule bacteria. Intensive-growing strains of B. japonicum KB11 and KC19 had identical set of fragments and formed rpoB type HI. Strains with a slow growth of B. japonicum VKM B-1967, 46, KC23 were characterized by unique restriction profiles. Despite the presence of two common fragments of 525 and 160 bp in size, they are classified into different rpoB types (HII, HIII, and HIV, respectively).

Under processing of amplificates with restriction enzyme NdeII, strains with intense growth formed three DNA fragments of 500, 170 and 120 bp in size, they are combined into one rpoB type – NI. The slow-growing rhizobia of B. japonicum VKM B-1967 and B. japonicum 46 are classified as NII rpoB type with four fragments in restriction profiles. The largest number of fragments (6 units) was observed in a pattern of slow-growing strain of B. japonicum KC23 (NIII rpoB type).

Therefore, using RFLP analysis of rpoB gene, we revealed significant differences between the strains of soybean nodule bacteria with different growth rates. With the use of restriction enzymes, MspI, HaeIII and NdeII, intensive-growing strains were combined into one rpoB type, while the slow-growing strains were more heterogeneous, they were classified in two to three rpoB types (depending on restriction enzymes).

It should be noted that the results we have obtained regarding the heterogeneity of B. japonicum strains are confirmed by literary data. The authors pay attention to the high degree of polymorphism of the studied nodule bacteria by different taxonomic markers. Using genome fingerprint techniques, S. Sikora et al. found that soybean nodule bacteria, common in Croatian soils, have significant differences and belong to three genetic groups (Sikora and Redzepović, 2003). Significant diversity of soybean microsymbionts has been noted in Kenyan agrocenoses. The study of ITS region of nodule bacteria has allowed to classify them into thirteen ITS types. At the same time, only five ITS types representatives were dominant soybean microsymbionts (Wasike et al., 2009). Y. Saeki (Saeki, 2011) using different experimental approaches (denaturing gradient gel electrophoresis (DGGE), T-RLFP analysis, automated ribosomal intergenic spacer analysis (ARISA), 16S rDNA and 16S-23S rDNA sequences) described soil populations of soybean rhizobia in Japanese soils. It has been established that the diversity of local nodule bacteria depends on the geographical and climatic characteristics of the region, and also associated with the diversity of host plant genotypes. Our previous studies have shown that intensive-growing strains of B. japonicum KB11, KC19, KC22 belong to the genetic group USDA 123 by the structure of the intergenic region of 16S-23S rRNA (Krutylo and Zotov, 2015). According to many authors, the representatives of this gene group are characterized by increased saprophytic competence – they can live for a long time in the soil without the host plant (Madrzak et al., 1995; Godoy et al., 2008). Intensive-growing soybean rhizobia were also characterized by increased survival in the soil. The typical B. japonicum strain and the slow-growing strains that we have isolated are representatives of two genetic groups – USDA 6 (B. japonicum VKM B-1967, 46, КН10) and USDA 4 (B. japonicum КС23).

CONCLUSION

In our study we have shown that slow and intensive-growing soybean nodule bacteria, common in Ukrainian soils, differ significantly in phenotypic and genotypic features.

All studied rhizobia can form active symbiotic relationships with soybean. Upon interaction with Chinese cowpea, intensive-growing strains form an ineffective symbiosis with a low nitrogen fixation (phenotype Nod+Fix), while slow-growing strains form complete nitrogen fixing systems (phenotype Nod+Fix+).

Upon cultivation in the mannitol-yeast medium, B. japonicum strains are capable of synthesizing phytohormonal substances of auxin, cytokinin and gibberellic nature. Intensive-growing strain of B. japonicum KC19 synthesizes a large number of auxins and cytokinins, while the slow-growing strain of B. japonicum KC23 is significantly superior by the amount of gibberellins.

According to the results of REP-PCR, the studied strains are classified into two separate groups of nodule bacteria, which corresponds to their division into groups by the growth rate (slow and intense growth).

The RFLP analysis of rpoB gene revealed significant differences between B. japonicum strains with different growth rates. Under the use of MspI, HaeIII and NdeII restriction enzymes, intensive-growing strains were combined into one rpoB type, while the slow-growing strains were more heterogeneous and classified as two to three rpoB types.

Acknowledgment: We are grateful to the Head of the laboratory of microbiological monitoring and bioremediation of soils of the All-Russian Research Institute of Agricultural Microbiology, Candidate of Biology E.E. Andronov for his assistance in conducting the restriction analysis, discussion of the obtained results, and valuable advice.

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