Distribution and Characterization of the Indigenous Soybean-Nodulating Bradyrhizobia in the Philippines

*Maria Luisa Tabing Mason and Yuichi Saeki*

#### **Abstract**

The research about the indigenous soybean bradyrhizobia in the Philippines is scarce, and this greatly influences the improvement of soybean production in the country. Thus, soil samples were collected from 11 locations in the country and were used to isolate the indigenous bradyrhizobia in the soil. Through the use of polymerase chain reaction—restriction fragment length polymorphism (PCR-RFLP) and sequence analysis of the 16S rRNA gene, 16S-23S rRNA gene internal transcribed spacer (ITS) region and *rpoB* housekeeping gene, the most abundant and dominant indigenous bradyrhizobia in the country were identified. Then, the representative isolates of the most dominant species per location were used to test their symbiotic efficiency and N-fixation ability with the local soybean cultivars. The results showed that among all the tested indigenous strains, the *B. elkanii* IS-2 is the most effective and efficient microsymbiont of the Philippines' local soybean cultivars. This report was able to provide necessary information on the distribution of soybean bradyrhizobia in the Philippines and characterized the symbiotic performance of the indigenous strains.

**Keywords:** tropical rhizobia, genomic diversity, N-fixation, symbiotic performance

#### **1. Introduction**

Soybean (*Glycine max* [L.]) is a leguminous plant that can form a symbiotic relationship with the nitrogen-fixing group of bacteria living in the rhizosphere, which are generally termed as rhizobia. In the Philippines, soybean production has been limited by the poor grain yield which leads to the importation of more than 90% of the country's demand. Thus, it is essential to look for an alternative way to increase the volume of production per unit area.

The research about tropical bradyrhizobia indicated a high diversity of species and their distribution has been reported to be due to several abiotic and biotic factors such as soil acidity [1–3], alkalinity [3, 4], temperature [1, 5–11], climate [12, 13], soil water status [14, 15], soil type [2, 14, 16–18], and soil management or cultural practices [2, 14, 19–22]. In case of the Philippines, the pioneer research that was able to identify the most dominant species of bradyrhizobia in the country reported that *B. elkanii* species was the most abundant, followed by the

*B. diazoefficiens*, *B. japonicum*, and some yet unclassified *Bradyrhizobium* sp. [14]. In this later study, it was identified that the distribution of these indigenous species of bradyrhizobia were influenced mainly by the water status of the soil, followed by soil pH, nutrient content, and soil type.

Previous studies have reported that aside from the various agro-environmental factors, the competition with the native rhizobia is a hindrance for a successful inoculation [23, 24]. The utilization of inoculants for legumes had shown promising results for the increase in grain yield as evidenced by recent reports [25, 26]. The role of the biological nitrogen fixation (BNF) in providing the N requirement of the plant in a natural way has been deemed necessary especially these times that the soil has become more degraded due to over-fertilization. The indiscriminate use of NPK fertilizer could cause soil pollution and less crop production [27]. Therefore, it is essential to select and evaluate the symbiotic competitiveness of the indigenous strains which are native and existing in high density in the country. The use of different genetic markers to accurately identify the rhizobia for taxonomic purposes has been proposed [28] and so we have used three genetic markers such as the 16S rRNA gene, 16S-23S rRNA gene internal transcribed spacer (ITS) region, and the *rpoB* housekeeping gene.

Thus, this study was formulated with the aim to utilize the recently identified indigenous bradyrhizobia in the Philippines and characterize their symbiotic performance with the local soybean cultivars.

#### **2. Materials and methods**

#### **2.1 Collection of soil and soybean cultivation**

The soil samples were collected from 11 locations in the Philippines, where some basic information on the sites are listed in **Table 1**. The collection of soil was conducted by first removing the surface litters then, obtaining a bar of soil with a dimension of approximately 20 cm in depth and 3 cm in thickness that weighs about 1 kg. A total of 10 subsamples per location were obtained and were mixed thoroughly until a 1 kg of composite soil sample was taken. A 0.5 kg soil was air-dried for the chemical analyses while the remaining 0.5 kg of the fresh soil was used for the soybean cultivation.

**157**

*Distribution and Characterization of the Indigenous Soybean-Nodulating Bradyrhizobia…*

distilled water until the initial weight of the pot was reached.

**2.2 Isolation of soybean rhizobia and DNA extraction**

The cultivation of soybean was performed using a 1-L capacity culture pots (n = 3). Each pot was filled with vermiculite and a N-free solution [29] was added at 40% (vol/vol) water content. The culture pots were sterilized by autoclaving for 20 min at 121°C. Meanwhile, the soybean seeds were surface-sterilized by soaking into a 70% EtOh for 30 s, then by a diluted sodium hypochlorite solution (0.25% available chlorine) for 3 min and followed by washing with sterile distilled water for about 6–8 times. Then, a 2–3 g of soil sample was placed on the vermiculite at a depth of about 2–3 cm, the seeds were sown on the soil and the pot was weighed and recorded. The plants were grown inside a growth chamber for 28 days at 28°C (8 h, night) and 33°C (16 h, day) then were supplied weekly with sterile

After 28 days, approximately 20 random nodules were collected from the roots of each soybean plants and were sterilized with 70% EtOh and sodium hypochlorite solution as previously described [29]. Each nodule was homogenized with sterile distilled water in a microtube and streaked on to a yeast-extract mannitol agar (YMA) plate [30]. The YMA plate was incubated in the dark at 28°C for about 1 week until a single colony was formed. After then, the single colony was streaked on to a YMA plate containing a 0.002% (wt/wt) bromothymol blue (BTB) [31] and was incubated as above. Repeated streaking was done until a pure single colony was obtained which was cultured for about 3–4 days in a HEPES-MES (HM) broth culture [32, 33] at 28°C in a shaker for 120 rpm. After then, the bacteria cells were collected by centrifugation at 9000×*g* and washed with sterile distilled water. The DNA was extracted by using BL buffer as described [34] from the method reported by Hiraishi et al. [35].

**2.3 PCR amplification of the 16S rRNA gene, ITS region, and rpoB gene**

annealing periods which were conducted at 30 s for each step.

set at 72°C for 5 min and indefinite preservation at 4°C.

**ITS region, and rpoB gene**

For the *rpoB* gene, simplification was done using the following primer sets: *rpo*B83F: 5′-CCTSATCGAGGTTCAC AGAAGGC-3′ and *rpo*B1540R:

5′-AGCTGCGAGGAACCGAAG-3′ [37]. The PCR cycle conditions were as follows: pre-run at 94°C for 5 min; followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 1 min, and extension at 72°C for 1 min. Final extension was

**2.4 Restriction fragment length polymorphism (RFLP) of the 16S rRNA gene,** 

The successfully amplified products were subjected to the RFLP treatment using four restriction enzymes which were *HhaI*, *HaeIII*, *MspI*, and *XspI*. For the rpoB gene, the enzymes that were used for RFLP are *HaeIII*, *MspI*, and *AluI*.

For the amplification of the 16S rRNA gene, the primer set: 16S-F: 5′ AGAG TTTGATCCTGGCTCAG-3′ and 16S-R2: 5′- CGGCTACCTTGTTACGACTT-3′ [36]. The PCR tubes were then placed in the PCR Thermal Cycler (TaKaRa Co. Ltd.) with the following conditions: pre-run at 94°C for 5 min; followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. Final extension was set at 72°C for 10 min and indefinite preservation at 4°C. On the other hand, the PCR amplification of the ITS region was conducted using the following primer set: Bra-ITS-F: 5-GACTGGGGTGAAGTCGTAAC-3′ and Bra-ITS-R1: 5′-ACGTCCTTCATCGCC TC-3′ [6]. The PCR cycle for the ITS region was almost the same with the 16S rRNA gene except for a shorter denaturation and

*DOI: http://dx.doi.org/10.5772/intechopen.85709*

#### **Table 1.**

*Result of the soil chemical analysis on the 11 locations in the Philippines.*

*a*

*b*

*Distribution and Characterization of the Indigenous Soybean-Nodulating Bradyrhizobia… DOI: http://dx.doi.org/10.5772/intechopen.85709*

The cultivation of soybean was performed using a 1-L capacity culture pots (n = 3). Each pot was filled with vermiculite and a N-free solution [29] was added at 40% (vol/vol) water content. The culture pots were sterilized by autoclaving for 20 min at 121°C. Meanwhile, the soybean seeds were surface-sterilized by soaking into a 70% EtOh for 30 s, then by a diluted sodium hypochlorite solution (0.25% available chlorine) for 3 min and followed by washing with sterile distilled water for about 6–8 times. Then, a 2–3 g of soil sample was placed on the vermiculite at a depth of about 2–3 cm, the seeds were sown on the soil and the pot was weighed and recorded. The plants were grown inside a growth chamber for 28 days at 28°C (8 h, night) and 33°C (16 h, day) then were supplied weekly with sterile distilled water until the initial weight of the pot was reached.

#### **2.2 Isolation of soybean rhizobia and DNA extraction**

After 28 days, approximately 20 random nodules were collected from the roots of each soybean plants and were sterilized with 70% EtOh and sodium hypochlorite solution as previously described [29]. Each nodule was homogenized with sterile distilled water in a microtube and streaked on to a yeast-extract mannitol agar (YMA) plate [30]. The YMA plate was incubated in the dark at 28°C for about 1 week until a single colony was formed. After then, the single colony was streaked on to a YMA plate containing a 0.002% (wt/wt) bromothymol blue (BTB) [31] and was incubated as above. Repeated streaking was done until a pure single colony was obtained which was cultured for about 3–4 days in a HEPES-MES (HM) broth culture [32, 33] at 28°C in a shaker for 120 rpm. After then, the bacteria cells were collected by centrifugation at 9000×*g* and washed with sterile distilled water. The DNA was extracted by using BL buffer as described [34] from the method reported by Hiraishi et al. [35].

#### **2.3 PCR amplification of the 16S rRNA gene, ITS region, and rpoB gene**

For the amplification of the 16S rRNA gene, the primer set: 16S-F: 5′ AGAG TTTGATCCTGGCTCAG-3′ and 16S-R2: 5′- CGGCTACCTTGTTACGACTT-3′ [36]. The PCR tubes were then placed in the PCR Thermal Cycler (TaKaRa Co. Ltd.) with the following conditions: pre-run at 94°C for 5 min; followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. Final extension was set at 72°C for 10 min and indefinite preservation at 4°C.

On the other hand, the PCR amplification of the ITS region was conducted using the following primer set: Bra-ITS-F: 5-GACTGGGGTGAAGTCGTAAC-3′ and Bra-ITS-R1: 5′-ACGTCCTTCATCGCC TC-3′ [6]. The PCR cycle for the ITS region was almost the same with the 16S rRNA gene except for a shorter denaturation and annealing periods which were conducted at 30 s for each step.

For the *rpoB* gene, simplification was done using the following primer sets: *rpo*B83F: 5′-CCTSATCGAGGTTCAC AGAAGGC-3′ and *rpo*B1540R: 5′-AGCTGCGAGGAACCGAAG-3′ [37]. The PCR cycle conditions were as follows: pre-run at 94°C for 5 min; followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 1 min, and extension at 72°C for 1 min. Final extension was set at 72°C for 5 min and indefinite preservation at 4°C.

#### **2.4 Restriction fragment length polymorphism (RFLP) of the 16S rRNA gene, ITS region, and rpoB gene**

The successfully amplified products were subjected to the RFLP treatment using four restriction enzymes which were *HhaI*, *HaeIII*, *MspI*, and *XspI*. For the rpoB gene, the enzymes that were used for RFLP are *HaeIII*, *MspI*, and *AluI*.

*Nitrogen Fixation*

soil pH, nutrient content, and soil type.

performance with the local soybean cultivars.

**2.1 Collection of soil and soybean cultivation**

*Result of the soil chemical analysis on the 11 locations in the Philippines.*

**2. Materials and methods**

*B. diazoefficiens*, *B. japonicum*, and some yet unclassified *Bradyrhizobium* sp. [14]. In this later study, it was identified that the distribution of these indigenous species of bradyrhizobia were influenced mainly by the water status of the soil, followed by

Previous studies have reported that aside from the various agro-environmental factors, the competition with the native rhizobia is a hindrance for a successful inoculation [23, 24]. The utilization of inoculants for legumes had shown promising results for the increase in grain yield as evidenced by recent reports [25, 26]. The role of the biological nitrogen fixation (BNF) in providing the N requirement of the plant in a natural way has been deemed necessary especially these times that the soil has become more degraded due to over-fertilization. The indiscriminate use of NPK fertilizer could cause soil pollution and less crop production [27]. Therefore, it is essential to select and evaluate the symbiotic competitiveness of the indigenous strains which are native and existing in high density in the country. The use of different genetic markers to accurately identify the rhizobia for taxonomic purposes has been proposed [28] and so we have used three genetic markers such as the 16S rRNA gene, 16S-23S rRNA gene

internal transcribed spacer (ITS) region, and the *rpoB* housekeeping gene.

Thus, this study was formulated with the aim to utilize the recently identified indigenous bradyrhizobia in the Philippines and characterize their symbiotic

The soil samples were collected from 11 locations in the Philippines, where some basic information on the sites are listed in **Table 1**. The collection of soil was conducted by first removing the surface litters then, obtaining a bar of soil with a dimension of approximately 20 cm in depth and 3 cm in thickness that weighs about 1 kg. A total of 10 subsamples per location were obtained and were mixed thoroughly until a 1 kg of composite soil sample was taken. A 0.5 kg soil was air-dried for the chemical analyses while the remaining 0.5 kg of the fresh soil was used for the soybean cultivation.

**156**

*a*

*b This study* **Table 1.**

*Mason et al., 2018*

The reference strains that were used in this study are the *Bradyrhizobium* USDA strains (*B. japonicum* USDA 4, 6<sup>T</sup> , 38, 62, 115, 122, 123, 124, 125, 127, 129, 135, *B. diazoefficiens* USDA 110<sup>T</sup> , *B. elkanii* 31, 46, 61, 76<sup>T</sup> , 94, 130, and *B. liaoningense* 3622<sup>T</sup> ) which were previously described [38]. This was done in a 10-μL reaction mixture containing a 2.5-μL amplified PCR product and was incubated in a 37°C for 16 h. Afterward, a 3–4% agarose gel was used in a submerged gel electrophoresis for about 60 min, stained with ethidium bromide and the patterns were visualized using a Luminiscent Image Analyzer LAS-4000 (FUJIFILM Tokyo, Japan).

#### **2.5 Single-strain inoculation test**

After the amplification and the RFLP treatment of the 16S rRNA gene, a singlestrain inoculation test was conducted for all the amplified isolates that shared the same restriction enzymes' fragment patterns with the USDA *Bradyrhizobium* reference strains. This was done to confirm the strain's capability to nodulate soybean and was tested on two local varieties which are the PSB-SY2 and Collection 1 which are both commercially available across the country.

The cultivation of soybean was conducted as described above, but without soil. Each isolate was cultured in a YM broth (YMB) [30] at 28°C for about 1 week on a shaker. After then, the cultures were diluted with sterile distilled water at about 106 cells mL<sup>−</sup><sup>1</sup> and were inoculated on the cultivated soybean at a rate of 1.0 mL per seed. This was done with three replications. After inoculation, the weight of the pot was recorded and it was placed inside a growth chamber with a condition set to mimic the average temperature in the Philippines at 26°C (8 h, night) and 33°C (16 h, day). The same condition was used for the cultivation of an uninoculated control and a positive control pot that was inoculated with *B. diazoefficiens* USDA110. The pots were kept inside the growth chamber for 28 days and were supplied weekly with sterile distilled water until the initial weight of each pot was reached.

#### **2.6 Sequence analysis of the 16S rRNA gene and ITS region**

According to the similarities of the band patterns through the RFLP treatment, a representative of the most abundant isolates was chosen for each location. In total, there were 11 isolates that were selected to confirm the nucleotide sequence of the 16S rRNA gene and the ITS region. The sequence primers that were used were reported previously [22]. From the PCR amplified product, the samples were purified according to the protocol of the manufacturer (Nucleospin® Gel and PCR Clean-up; Macherey-Nagel, Germany). Then, the samples were sent to the company for the sequence analysis (Eurofins Genomics, Tokyo, Japan).

Then, the Basic Local Alignment Search Tool (BLAST) program in DNA Databank of Japan (DDBJ) was used to determine the nucleotide homology of the isolates. Only the sequences with a similarity of at least 99% for the 16S rRNA and 96% for the ITS region with our isolates were retrieved from the BLAST database. The alignment was performed using the ClustalW and Neighbor-Joining [21] method was used to construct the phylogenetic trees. The genetic distances were computed using the Kimura 2-parameter model [39] in the Molecular Evolutionary Genetic Analysis (MEGA v7) software [40]. Subsequently, the phylogenetic trees were bootstrapped with 1000 replications. All the nucleotide sequences determined in this study were deposited in DDBJ at http://www.ddbj.nig.ac.jp/.

**159**

**Table 2.**

*Distribution and Characterization of the Indigenous Soybean-Nodulating Bradyrhizobia…*

**3.1 Soil analysis and characterization of the indigenous bradyrhizobia**

The soil samples that were used in this study were all slightly to moderately acidic (5.22–6.64) with non-saline condition (0.05–0.20 dS/m), low nutrient status as evidenced by low amounts of NPK and CEC (**Table 1**). These values are generally typical of agricultural soils that are used for crop production all throughout the year. These results showed that the soils used in this study have low fertility status

The growth morphologies of the pure single colony for each strain of bradyrhizobia were characterized and listed in **Table 2**. All the isolates were slow growers which were able to form single colonies measuring about 2 mm between 5 and 7 days upon streaking on YMA plates and incubation in a dark room. Based on the morphology, the isolates were grouped into three. Group I include the isolates IS-2, NE1–6, NR-2, and BO-4 which were translucent and the colonies are circular

*Characterization of the morphology of the indigenous bradyrhizobia isolated from Philippines' soil according to* 

*their growth on Yeast-Extract Mannitol Agar plate medium [30].*

*DOI: http://dx.doi.org/10.5772/intechopen.85709*

that indicated the need for soil restoration strategies.

**3. Results**

*Distribution and Characterization of the Indigenous Soybean-Nodulating Bradyrhizobia… DOI: http://dx.doi.org/10.5772/intechopen.85709*

#### **3. Results**

*Nitrogen Fixation*

*gense* 3622<sup>T</sup>

106

reached.

cells mL<sup>−</sup><sup>1</sup>

strains (*B. japonicum* USDA 4, 6<sup>T</sup>

*B. diazoefficiens* USDA 110<sup>T</sup>

(FUJIFILM Tokyo, Japan).

**2.5 Single-strain inoculation test**

are both commercially available across the country.

**2.6 Sequence analysis of the 16S rRNA gene and ITS region**

for the sequence analysis (Eurofins Genomics, Tokyo, Japan).

The reference strains that were used in this study are the *Bradyrhizobium* USDA

, *B. elkanii* 31, 46, 61, 76<sup>T</sup>

reaction mixture containing a 2.5-μL amplified PCR product and was incubated in a 37°C for 16 h. Afterward, a 3–4% agarose gel was used in a submerged gel electrophoresis for about 60 min, stained with ethidium bromide and the patterns were visualized using a Luminiscent Image Analyzer LAS-4000

After the amplification and the RFLP treatment of the 16S rRNA gene, a singlestrain inoculation test was conducted for all the amplified isolates that shared the same restriction enzymes' fragment patterns with the USDA *Bradyrhizobium* reference strains. This was done to confirm the strain's capability to nodulate soybean and was tested on two local varieties which are the PSB-SY2 and Collection 1 which

The cultivation of soybean was conducted as described above, but without soil. Each isolate was cultured in a YM broth (YMB) [30] at 28°C for about 1 week on a shaker. After then, the cultures were diluted with sterile distilled water at about

According to the similarities of the band patterns through the RFLP treatment, a representative of the most abundant isolates was chosen for each location. In total, there were 11 isolates that were selected to confirm the nucleotide sequence of the 16S rRNA gene and the ITS region. The sequence primers that were used were reported previously [22]. From the PCR amplified product, the samples were purified according to the protocol of the manufacturer (Nucleospin® Gel and PCR Clean-up; Macherey-Nagel, Germany). Then, the samples were sent to the company

Then, the Basic Local Alignment Search Tool (BLAST) program in DNA Databank of Japan (DDBJ) was used to determine the nucleotide homology of the isolates. Only the sequences with a similarity of at least 99% for the 16S rRNA and 96% for the ITS region with our isolates were retrieved from the BLAST database. The alignment was performed using the ClustalW and Neighbor-Joining [21] method was used to construct the phylogenetic trees. The genetic distances were computed using the Kimura 2-parameter model [39] in the Molecular Evolutionary Genetic Analysis (MEGA v7) software [40]. Subsequently, the phylogenetic trees were bootstrapped with 1000 replications. All the nucleotide sequences determined in this study were deposited in DDBJ at

per seed. This was done with three replications. After inoculation, the weight of the pot was recorded and it was placed inside a growth chamber with a condition set to mimic the average temperature in the Philippines at 26°C (8 h, night) and 33°C (16 h, day). The same condition was used for the cultivation of an uninoculated control and a positive control pot that was inoculated with *B. diazoefficiens* USDA110. The pots were kept inside the growth chamber for 28 days and were supplied weekly with sterile distilled water until the initial weight of each pot was

and were inoculated on the cultivated soybean at a rate of 1.0 mL

) which were previously described [38]. This was done in a 10-μL

, 38, 62, 115, 122, 123, 124, 125, 127, 129, 135,

, 94, 130, and *B. liaonin-*

**158**

http://www.ddbj.nig.ac.jp/.

#### **3.1 Soil analysis and characterization of the indigenous bradyrhizobia**

The soil samples that were used in this study were all slightly to moderately acidic (5.22–6.64) with non-saline condition (0.05–0.20 dS/m), low nutrient status as evidenced by low amounts of NPK and CEC (**Table 1**). These values are generally typical of agricultural soils that are used for crop production all throughout the year. These results showed that the soils used in this study have low fertility status that indicated the need for soil restoration strategies.

The growth morphologies of the pure single colony for each strain of bradyrhizobia were characterized and listed in **Table 2**. All the isolates were slow growers which were able to form single colonies measuring about 2 mm between 5 and 7 days upon streaking on YMA plates and incubation in a dark room. Based on the morphology, the isolates were grouped into three. Group I include the isolates IS-2, NE1–6, NR-2, and BO-4 which were translucent and the colonies are circular


**Table 2.**

*Characterization of the morphology of the indigenous bradyrhizobia isolated from Philippines' soil according to their growth on Yeast-Extract Mannitol Agar plate medium [30].*

in shape with slightly convex elevation and an entire margin. When they were manipulated with a needle, the colony was liquid. Group II include the isolates BA-24, SO-1, LT-3, and SK-5 were translucent with circular colonies, convex elevation with entire margin. When manipulated with a needle, the colonies have mucoid viscosity. On the other hand, last group (III) are the isolates GI-4 and NE2-37 which have similar growth morphology with Group II except that their viscosity was intermediate between liquid and mucoid. All the isolates produced alkaline substances when grown on YMA plate with BTB which is an indication of the *Bradyrhizobium* genus.

#### **Figure 1.**

*Phylogenetic tree based on the sequence analysis of the 16S rRNA gene. The tree was constructed using the Neighbor-Joining method with the Kimura 2-parameter (K2P) distance correlation model and 1000 bootstrap replications in MEGA v.7 software. The accession numbers are indicated only for sequences obtained from BLAST. The isolates in this study are indicated with letters and number combinations, for example: BO-4– isolate no. 4 collected from Bohol.*

**161**

**Figure 2.**

*Distribution and Characterization of the Indigenous Soybean-Nodulating Bradyrhizobia…*

rhizobia in the Philippines are classified under the genus *Bradyrhizobium*, and are separated into its two species, *B. japonicum* and *B. elkanii*, according to the

As seen in **Figure 1**, it is evident that the 11 most abundant indigenous soybean

*Phylogenetic tree based on the sequence analysis of the 16S-23S rRNA internal transcribed spacer (ITS) region. The tree was constructed using the Neighbor-Joining method with the Kimura 2-parameter (K2P) distance correlation model and 1000 bootstrap replications in MEGA v.7 software. The accession numbers are indicated only for sequences obtained from BLAST. The isolates in this study are indicated with letters and number* 

*combinations, for example: BO-4–isolate no. 4 collected from Bohol.*

*DOI: http://dx.doi.org/10.5772/intechopen.85709*

**3.2 Distribution of indigenous soybean bradyrhizobia**

*Distribution and Characterization of the Indigenous Soybean-Nodulating Bradyrhizobia… DOI: http://dx.doi.org/10.5772/intechopen.85709*

#### **3.2 Distribution of indigenous soybean bradyrhizobia**

*Nitrogen Fixation*

the *Bradyrhizobium* genus.

in shape with slightly convex elevation and an entire margin. When they were manipulated with a needle, the colony was liquid. Group II include the isolates BA-24, SO-1, LT-3, and SK-5 were translucent with circular colonies, convex elevation with entire margin. When manipulated with a needle, the colonies have mucoid viscosity. On the other hand, last group (III) are the isolates GI-4 and NE2-37 which have similar growth morphology with Group II except that their viscosity was intermediate between liquid and mucoid. All the isolates produced alkaline substances when grown on YMA plate with BTB which is an indication of

*Phylogenetic tree based on the sequence analysis of the 16S rRNA gene. The tree was constructed using the Neighbor-Joining method with the Kimura 2-parameter (K2P) distance correlation model and 1000 bootstrap replications in MEGA v.7 software. The accession numbers are indicated only for sequences obtained from BLAST. The isolates in this study are indicated with letters and number combinations, for example: BO-4–*

**160**

**Figure 1.**

*isolate no. 4 collected from Bohol.*

As seen in **Figure 1**, it is evident that the 11 most abundant indigenous soybean rhizobia in the Philippines are classified under the genus *Bradyrhizobium*, and are separated into its two species, *B. japonicum* and *B. elkanii*, according to the

**Figure 2.**

*Phylogenetic tree based on the sequence analysis of the 16S-23S rRNA internal transcribed spacer (ITS) region. The tree was constructed using the Neighbor-Joining method with the Kimura 2-parameter (K2P) distance correlation model and 1000 bootstrap replications in MEGA v.7 software. The accession numbers are indicated only for sequences obtained from BLAST. The isolates in this study are indicated with letters and number combinations, for example: BO-4–isolate no. 4 collected from Bohol.*

phylogenetic tree from the sequence analysis of the 16S rRNA gene. To further confirm the classification of the indigenous bradyrhizobia, the phylogenetic trees constructed from the ITS region and the *rpoB* gene are presented in **Figures 2** and **3**, respectively. For the ITS region and the *rpoB* gene, the isolates were distinctly grouped into three species, *B. elkanii*, *B. japonicum*, and *B. diazoefficiens*. Additionally, an independent cluster composed of the representative isolates GI-4

#### **Figure 3.**

*Phylogenetic tree based on the sequence analysis of the rpoB housekeeping gene. The tree was constructed using the Neighbor-Joining method with the Kimura 2-parameter (K2P) distance correlation model and 1000 bootstrap replications in MEGA v.7 software. The accession numbers are indicated only for sequences obtained from BLAST. The isolates in this study are indicated with letters and number combinations, for example: BO-4–isolate no.4 collected from Bohol.*

**163**

**Table 3.**

*housekeeping gene.*

*Distribution and Characterization of the Indigenous Soybean-Nodulating Bradyrhizobia…*

and NE2–37 that are seen in the ITS region and *rpoB* phylogenetic trees were treated as *Bradyrhizobium* sp. due to their nucleotide divergence with the known species

Meanwhile, the distribution of the most abundant soybean bradyrhizobia in the country is shown in **Table 3**, which was classified according to the results of the sequence analysis of the three genetic markers used in this study. From here, it can be seen that 4 of the 11 locations were dominated with *B. elkanii* species (37.74%), 3 locations were dominated by the isolates under the *B. diazoefficiens* (28.54%), whereas 2 locations each were dominated by the species of *B. japonicum* (16.98% and *Bradyrhizobium* sp. (16.74%). This indicated that in the Philippines, the species of *B. elkanii* is the most prevalent in terms of population and the most widespread in terms of location as its presence was detected even in minor populations on all

**3.3 Symbiotic efficiency and N-fixation ability of the indigenous bradyrhizobia**

*Percentage distribution of the dominant Bradyrhizobium species in the Philippines as identified from the sequence analysis of the 16S rRNA gene, 16S-23S internal transcribed spacer (ITS) region, and rpoB* 

Upon classification, it is important to determine the capability of the indigenous bradyrhizobia for their symbiotic performance and N-fixation ability. As can be seen in **Figure 4A**, although USDA110 strain has the highest N-fixation ability, it should be noted that the amount of N that was fixed by *B. elkanii* IS-2 is the highest among all the indigenous bradyrhizobia isolated from the Philippines' soil on *Rj*<sup>4</sup> plants. However, the N-fixation ability of IS-2 was comparably similar with other strains (GI-4, NE2–37, and SK-5) with the non-*Rj* plants. The lowest N-fixation ability was observed from the strain LT-3 which was classified under the *B. diazoefficiens* species. This suggested that the process of biological N-fixation is a mutual relationship that is influenced by both the plant and the rhizobia and that the plantrhizobia compatibility should be taken into consideration for inoculation strategies. Presented in **Figure 4B** is the nodulation test performed on the strains and it can be seen for *Rj*4 plants, there was not much significant difference in the nodulation

*DOI: http://dx.doi.org/10.5772/intechopen.85709*

the locations except for one, which was Sorsogon.

from the BLAST engine.

*Distribution and Characterization of the Indigenous Soybean-Nodulating Bradyrhizobia… DOI: http://dx.doi.org/10.5772/intechopen.85709*

and NE2–37 that are seen in the ITS region and *rpoB* phylogenetic trees were treated as *Bradyrhizobium* sp. due to their nucleotide divergence with the known species from the BLAST engine.

Meanwhile, the distribution of the most abundant soybean bradyrhizobia in the country is shown in **Table 3**, which was classified according to the results of the sequence analysis of the three genetic markers used in this study. From here, it can be seen that 4 of the 11 locations were dominated with *B. elkanii* species (37.74%), 3 locations were dominated by the isolates under the *B. diazoefficiens* (28.54%), whereas 2 locations each were dominated by the species of *B. japonicum* (16.98% and *Bradyrhizobium* sp. (16.74%). This indicated that in the Philippines, the species of *B. elkanii* is the most prevalent in terms of population and the most widespread in terms of location as its presence was detected even in minor populations on all the locations except for one, which was Sorsogon.

#### **3.3 Symbiotic efficiency and N-fixation ability of the indigenous bradyrhizobia**

Upon classification, it is important to determine the capability of the indigenous bradyrhizobia for their symbiotic performance and N-fixation ability. As can be seen in **Figure 4A**, although USDA110 strain has the highest N-fixation ability, it should be noted that the amount of N that was fixed by *B. elkanii* IS-2 is the highest among all the indigenous bradyrhizobia isolated from the Philippines' soil on *Rj*<sup>4</sup> plants. However, the N-fixation ability of IS-2 was comparably similar with other strains (GI-4, NE2–37, and SK-5) with the non-*Rj* plants. The lowest N-fixation ability was observed from the strain LT-3 which was classified under the *B. diazoefficiens* species. This suggested that the process of biological N-fixation is a mutual relationship that is influenced by both the plant and the rhizobia and that the plantrhizobia compatibility should be taken into consideration for inoculation strategies.

Presented in **Figure 4B** is the nodulation test performed on the strains and it can be seen for *Rj*4 plants, there was not much significant difference in the nodulation


#### **Table 3.**

*Nitrogen Fixation*

**162**

**Figure 3.**

*BO-4–isolate no.4 collected from Bohol.*

*Phylogenetic tree based on the sequence analysis of the rpoB housekeeping gene. The tree was constructed using the Neighbor-Joining method with the Kimura 2-parameter (K2P) distance correlation model and 1000 bootstrap replications in MEGA v.7 software. The accession numbers are indicated only for sequences obtained from BLAST. The isolates in this study are indicated with letters and number combinations, for example:* 

phylogenetic tree from the sequence analysis of the 16S rRNA gene. To further confirm the classification of the indigenous bradyrhizobia, the phylogenetic trees constructed from the ITS region and the *rpoB* gene are presented in **Figures 2** and **3**, respectively. For the ITS region and the *rpoB* gene, the isolates were distinctly grouped into three species, *B. elkanii*, *B. japonicum*, and *B. diazoefficiens*. Additionally, an independent cluster composed of the representative isolates GI-4

*Percentage distribution of the dominant Bradyrhizobium species in the Philippines as identified from the sequence analysis of the 16S rRNA gene, 16S-23S internal transcribed spacer (ITS) region, and rpoB housekeeping gene.*

**165**

*Distribution and Characterization of the Indigenous Soybean-Nodulating Bradyrhizobia…*

obtained the highest number of nodules for the non-*Rj* plants.

not be directly influenced by the *Rj* genotype of the plant.

ability of the strains, except for the low nodulation ability that was observed for the BO-4. In contrast, a significant difference in the nodulation ability was detected on the strains upon inoculation on the non-*Rj* plants. Although all the strains were able to form nodules on both soybean cultivars, the strains GI-4, NR-2, and SK-5

On the other hand, the symbiotic efficiency of the strains used in this study is presented in **Figure 4C**. Similar with the N-fixation ability, the USDA110 still possesses the highest symbiotic efficiency. But among all the indigenous bradyrhizobia, the strain IS-2 obtained the highest efficiency regardless of the *Rj* genotype of the soybean plants. As with the N-fixation, LT-3 obtained the least efficiency for symbiosis. This result indicated that the symbiotic efficiency of the rhizobia might

**4.1 Distribution and characterization of bradyrhizobia in the Philippines**

were reported to be dominated by species of *B. japonicum* and *B. diazoefficiens* [6, 9–11, 13, 45]. Our report showed that the distribution of bradyrhizobia in a tropical region like the Philippines seemed to be different from those of temperate regions. Meanwhile, it was included in a recent report that the distribution and abundance of *B. diazoefficiens* and *B. japonicum* at specific locations were due to the longer period of flooding conditions [14]. The effect of nutrient content and soil type were also correlated with the abundance of these two species. In a report by Shiina et al. [17], it was stated that the predominance of *B. diazoefficiens* was observed on more anaerobic condition; whereas, *B. japonicum* was predominant on aerobic soils which was supported by another study [18]. Additionally, it was reported that *B. diazoefficiens* becomes predominant with enhanced flooding condition [15]. These results confirmed that our observations for the abundant of *B. diazoefficiens*, followed by *B. japonicum* and *Bradyrhizobium* sp. on flooded areas in the country

**4.2 Symbiotic and N-fixation ability of indigenous bradyrhizobia**

In this report, the symbiotic performance, N-fixation and nodulation ability of the indigenous soybean bradyrhizobia form the Philippines were evaluated against that of the *B. diazoefficiens* USDA110 strain. The USDA110 has been extensively used in the world as a model strain for soybean inoculation due to its high ability for N-fixation and symbiotic efficiency [25, 46, 47]. Additionally, its possession of a complete set of denitrification genes that allows the release of N2 back into the atmosphere makes it an ideal strain also for climate change mitigation studies [17, 48–50].

The distribution of the most dominant and abundant species of soybean bradyrhizobia in the Philippines are reported in this study along with the characterization of their growth morphology. According to our earlier reports, we have elucidated that the Philippines was dominated by the soybean-nodulating bradyrhizobia that were classified under the *B. elkanii* species and the most important agro-environmental factors that affected their diversity and prevalence in the country was the similarity of soil pH, salinity, and temperature in the study locations [5, 14]. Our observation that there are abundant and high diversity of indigenous bradyrhizobia in the Philippines is similar with previous reports in other sub-tropical and tropical regions [12, 25, 41–44]. The temperate regions of Japan and USA were studied in the past and

*DOI: http://dx.doi.org/10.5772/intechopen.85709*

which were usually used for planting rice.

**4. Discussion**

#### **Figure 4.**

*Characterization of the dominant indigenous Bradyrhizobium strains isolated from the 11 locations in the Philippines based on the (A) amount of Nitrogen fixed (B) nodulation ability and (C) symbiotic efficiency as influenced by the single-strain inoculation test against the reference strain B. diazoefficiens USDA110 for the two soybean cultivars from the Philippines. Different letters indicate a significant difference by Tukey's test at p > 0.05, n=3, bar=SE.*

*Distribution and Characterization of the Indigenous Soybean-Nodulating Bradyrhizobia… DOI: http://dx.doi.org/10.5772/intechopen.85709*

ability of the strains, except for the low nodulation ability that was observed for the BO-4. In contrast, a significant difference in the nodulation ability was detected on the strains upon inoculation on the non-*Rj* plants. Although all the strains were able to form nodules on both soybean cultivars, the strains GI-4, NR-2, and SK-5 obtained the highest number of nodules for the non-*Rj* plants.

On the other hand, the symbiotic efficiency of the strains used in this study is presented in **Figure 4C**. Similar with the N-fixation ability, the USDA110 still possesses the highest symbiotic efficiency. But among all the indigenous bradyrhizobia, the strain IS-2 obtained the highest efficiency regardless of the *Rj* genotype of the soybean plants. As with the N-fixation, LT-3 obtained the least efficiency for symbiosis. This result indicated that the symbiotic efficiency of the rhizobia might not be directly influenced by the *Rj* genotype of the plant.

#### **4. Discussion**

*Nitrogen Fixation*

**164**

**Figure 4.**

*p > 0.05, n=3, bar=SE.*

*Characterization of the dominant indigenous Bradyrhizobium strains isolated from the 11 locations in the Philippines based on the (A) amount of Nitrogen fixed (B) nodulation ability and (C) symbiotic efficiency as influenced by the single-strain inoculation test against the reference strain B. diazoefficiens USDA110 for the two soybean cultivars from the Philippines. Different letters indicate a significant difference by Tukey's test at* 

#### **4.1 Distribution and characterization of bradyrhizobia in the Philippines**

The distribution of the most dominant and abundant species of soybean bradyrhizobia in the Philippines are reported in this study along with the characterization of their growth morphology. According to our earlier reports, we have elucidated that the Philippines was dominated by the soybean-nodulating bradyrhizobia that were classified under the *B. elkanii* species and the most important agro-environmental factors that affected their diversity and prevalence in the country was the similarity of soil pH, salinity, and temperature in the study locations [5, 14]. Our observation that there are abundant and high diversity of indigenous bradyrhizobia in the Philippines is similar with previous reports in other sub-tropical and tropical regions [12, 25, 41–44]. The temperate regions of Japan and USA were studied in the past and were reported to be dominated by species of *B. japonicum* and *B. diazoefficiens* [6, 9–11, 13, 45]. Our report showed that the distribution of bradyrhizobia in a tropical region like the Philippines seemed to be different from those of temperate regions.

Meanwhile, it was included in a recent report that the distribution and abundance of *B. diazoefficiens* and *B. japonicum* at specific locations were due to the longer period of flooding conditions [14]. The effect of nutrient content and soil type were also correlated with the abundance of these two species. In a report by Shiina et al. [17], it was stated that the predominance of *B. diazoefficiens* was observed on more anaerobic condition; whereas, *B. japonicum* was predominant on aerobic soils which was supported by another study [18]. Additionally, it was reported that *B. diazoefficiens* becomes predominant with enhanced flooding condition [15]. These results confirmed that our observations for the abundant of *B. diazoefficiens*, followed by *B. japonicum* and *Bradyrhizobium* sp. on flooded areas in the country which were usually used for planting rice.

#### **4.2 Symbiotic and N-fixation ability of indigenous bradyrhizobia**

In this report, the symbiotic performance, N-fixation and nodulation ability of the indigenous soybean bradyrhizobia form the Philippines were evaluated against that of the *B. diazoefficiens* USDA110 strain. The USDA110 has been extensively used in the world as a model strain for soybean inoculation due to its high ability for N-fixation and symbiotic efficiency [25, 46, 47]. Additionally, its possession of a complete set of denitrification genes that allows the release of N2 back into the atmosphere makes it an ideal strain also for climate change mitigation studies [17, 48–50].

Therefore, we hypothesized that the indigenous isolates SO-1, LT-3, and SK-5, which were phylogenetically clustered under the USDA110 would also prove to be as effective N-fixer and efficient microsymbiont of soybean cultivars from the Philippines. However, our results indicated that the N-fixation ability and symbiotic efficiency of LT-3 and SO-1 were very low in comparison to the other indigenous isolates. For the low performance of these two isolates, it is hypothesized that the inherent ability of these strains to fix N and establish a symbiotic relationship with soybean is low. This could be explained by the fact that their nodulation ability was comparably similar with the other strains which possess higher N-fixation ability and symbiotic efficiency. In contrast, the isolate IS-2, which was clustered under the *B. elkanii* species, showed the highest symbiotic efficiency for both *Rj*-genotypes of the soybean cultivar used and the highest N-fixation ability for *Rj*4 plants. In a previous report, the *Rj* genes that could restrict the nodulation of soybean by some strains of bradyrhizobia was summarized [51] but in case of our present report, all the strains in this study were not restricted by the two *Rj*-plants that were used. This led us to consider that the low N-fixation and symbiotic performance of some strains were not due to the restrictions from the *Rj*-genotypes of the plants but could be attributed to the strains' intrinsic capabilities. These observations might explain the reason for low yield of soybean in the Philippines. It was reported that many strains of *B. elkanii* were relatively inefficient microsymbionts of soybean and can induce chlorosis in soybean plants [52]. In a previous report [53], the high temperatures in tropical regions can limit the nodulation which could explain the low soybean yield.

It was expected that the strains which were classified as *B. diazoefficiens* could provide a better symbiotic performance than the other strains that were collected. However, the data showed that *B. elkanii* might establish a better symbiosis with local soybean cultivars in the Philippines. This result is crucial in order to devise strategies on how to increase the local production of soybean by inoculation with the indigenous strains.

Upon considering these results with the N-fixation and symbiotic performance ability of the strains, the number of nodules that can be formed from the singlestrain inoculation does not seem to influence the amount of N that each strain can fix nor their symbiotic ability.

#### **5. Conclusion**

In this report, we have revealed that the distribution of tropical soybean bradyrhizobia seemed to be different than those of temperate bradyrhizobia in terms of population dominance of *B. elkanii* on higher temperature region like the Philippines. Additionally, it is proposed that for the Philippines, the most efficient N-fixer and symbiotically efficient species of bradyrhizobia would be *B. elkanii*. Yet, our results were made under the laboratory conditions only, so the results that were obtained here might not be as expected when done in field condition. For future research, utilization of more local soybean varieties with different soil types both in a controlled environment and on natural field condition would be beneficial to target the development of a site-specific and useful potential soybean inoculant. The data generated in this report would be beneficial for the augmentation of inoculation strategies in the country.

#### **Acknowledgements**

The authors would like to acknowledge the contributions of John Philip Tanay, Emmanuel Victor Buniao, Mary Joy Portin, and Maria Leah Sevilla of Central Luzon

**167**

**Author details**

Maria Luisa Tabing Mason1

Nueva Ecija, Philippines

provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

and Yuichi Saeki<sup>2</sup>

2 Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan

\*Address all correspondence to: yt-saeki@cc.miyazaki-u.ac.jp

1 College of Agriculture, Central Luzon State University, Science City of Munoz,

\*

*Distribution and Characterization of the Indigenous Soybean-Nodulating Bradyrhizobia…*

State University for their help on some laboratory experiments. This work was supported by the JSPS Grant-in-Aid for Scientific Research (KAKENHI Grant Number:

*DOI: http://dx.doi.org/10.5772/intechopen.85709*

The authors declare no conflict of interest.

18K05376).

**Conflict of interest**

*Distribution and Characterization of the Indigenous Soybean-Nodulating Bradyrhizobia… DOI: http://dx.doi.org/10.5772/intechopen.85709*

State University for their help on some laboratory experiments. This work was supported by the JSPS Grant-in-Aid for Scientific Research (KAKENHI Grant Number: 18K05376).

## **Conflict of interest**

*Nitrogen Fixation*

the indigenous strains.

**5. Conclusion**

**Acknowledgements**

fix nor their symbiotic ability.

Therefore, we hypothesized that the indigenous isolates SO-1, LT-3, and SK-5, which were phylogenetically clustered under the USDA110 would also prove to be as effective N-fixer and efficient microsymbiont of soybean cultivars from the Philippines. However, our results indicated that the N-fixation ability and symbiotic efficiency of LT-3 and SO-1 were very low in comparison to the other indigenous isolates. For the low performance of these two isolates, it is hypothesized that the inherent ability of these strains to fix N and establish a symbiotic relationship with soybean is low. This could be explained by the fact that their nodulation ability was comparably similar with the other strains which possess higher N-fixation ability and symbiotic efficiency. In contrast, the isolate IS-2, which was clustered under the *B. elkanii* species, showed the highest symbiotic efficiency for both *Rj*-genotypes of the soybean cultivar used and the highest N-fixation ability for *Rj*4 plants. In a previous report, the *Rj* genes that could restrict the nodulation of soybean by some strains of bradyrhizobia was summarized [51] but in case of our present report, all the strains in this study were not restricted by the two *Rj*-plants that were used. This led us to consider that the low N-fixation and symbiotic performance of some strains were not due to the restrictions from the *Rj*-genotypes of the plants but could be attributed to the strains' intrinsic capabilities. These observations might explain the reason for low yield of soybean in the Philippines. It was reported that many strains of *B. elkanii* were relatively inefficient microsymbionts of soybean and can induce chlorosis in soybean plants [52]. In a previous report [53], the high temperatures in tropical regions can limit the nodulation which could explain the low soybean yield. It was expected that the strains which were classified as *B. diazoefficiens* could provide a better symbiotic performance than the other strains that were collected. However, the data showed that *B. elkanii* might establish a better symbiosis with local soybean cultivars in the Philippines. This result is crucial in order to devise strategies on how to increase the local production of soybean by inoculation with

Upon considering these results with the N-fixation and symbiotic performance ability of the strains, the number of nodules that can be formed from the singlestrain inoculation does not seem to influence the amount of N that each strain can

In this report, we have revealed that the distribution of tropical soybean bradyrhizobia seemed to be different than those of temperate bradyrhizobia in terms of population dominance of *B. elkanii* on higher temperature region like the Philippines. Additionally, it is proposed that for the Philippines, the most efficient N-fixer and symbiotically efficient species of bradyrhizobia would be *B. elkanii*. Yet, our results were made under the laboratory conditions only, so the results that were obtained here might not be as expected when done in field condition. For future research, utilization of more local soybean varieties with different soil types both in a controlled environment and on natural field condition would be beneficial to target the development of a site-specific and useful potential soybean inoculant. The data generated in this report would be beneficial for the augmentation of inoculation strategies in the country.

The authors would like to acknowledge the contributions of John Philip Tanay, Emmanuel Victor Buniao, Mary Joy Portin, and Maria Leah Sevilla of Central Luzon

**166**

The authors declare no conflict of interest.

#### **Author details**

Maria Luisa Tabing Mason1 and Yuichi Saeki<sup>2</sup> \*

1 College of Agriculture, Central Luzon State University, Science City of Munoz, Nueva Ecija, Philippines

2 Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan

\*Address all correspondence to: yt-saeki@cc.miyazaki-u.ac.jp

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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(*Glycine max* L.) in different

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*Nitrogen Fixation*

*Bradyrhizobium* strains with outstanding symbiotic performance to increase soybean yields in Mozambique. Agriculture, Ecosystems and

[33] Sameshima R, Isawa T, Sadowsky MJ, Hamada T, Kasai H, Shutsrirung A, et al. Phylogeny and distribution of extra-slow-growing *Bradyrhizobium japonicum* harboring high copy numbers

[34] Minami M, Yamakawa T, Yamamoto

[35] Hiraishi A, Kamagata Y, Nakamura

amplification and restriction fragment length polymorphism analysis of 16S rRNA genes from methanogens.

Bioengineering. 1995;**79**:523-529. DOI: 10.1016/0922-338X(95)94742-A

[37] Martens M, Dawyndt P, Coopman R, Gillis M, De Vos P, Willems A. Advantages of multilocus sequence analysis for taxonomic studies: A case study using 10 housekeeping genes in the genus *Ensifer* (including former *Sinorhizobium*). International Journal of Systematic and Evolutionary Microbiology.

[38] Saeki Y, Aimi N, Hashimoto M, Tsukamoto S, Kaneko A, Yoshida N, et al. Grouping of *Bradyrhizobium* USDA strains by sequence analysis of 16S rDNA and 16S-23S rDNA internal transcribed spacer region. Soil Science and Plant Nutrition. 2004;**50**:517-525. DOI: 10.1080/00380768.2004.10408508

K. Polymerase chain reaction

Journal of Fermentation and

[36] Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology.

1991;**173**:697-703

2008;**58**:200-214

A, Akao S, Saeki Y. Estimation of nodulation tendency among *Rj*-genotype soybeans using the bradyrhizobial community isolated from an Andosol. Soil Science and Plant Nutrition. 2009;**55**:65-72. DOI: 10.1111/j.1747-0765.2008.00333.x

of RSα, RSβ and IS1631. FEMS Microbiology Ecology. 2003;**44**:191-202. DOI: 10.1016/ S0168-6496(03)00009-6

Environment. 2017;**246**:291-305. DOI:

[26] Heerwaarden J, Baijukya F, Kyei-Boahen S, Adjei-Nsiah S, Ebanyat P, Kamai N, et al. Soyabean response to rhizobium inoculation across sub-Saharan Africa: Patterns of variation and the role of promiscuity. Agriculture,

Ecosystems and Environment. 2018;**261**:211-218. DOI: 10.1016/j.

2014. 56pp. DOI: 10.5772/57287

[29] Saeki Y, Akagi I, Takaki H, Nagatomo Y. Diversity of indigenous *Bradyrhizobium* strains isolated from three different *Rj*-soybean cultivars in terms of randomly amplified polymorphic DNA and intrinsic antibiotic resistance. Soil Science and Plant Nutrition. 2000;**46**:917-926. DOI: 10.1080/00380768.2000.10409157

[30] Vincent JM. A Manual for the Practical Study of the Root-Nodule Bacteria. Oxford: Blackwell Scientific;

[31] Keyser HH, Bohlool BB, Hu TS, Weber DF. Fast-growing rhizobia isolated from root nodules of soybean.

[32] Cole MA, Elkan GH. Transmissible resistance to Penicillin-G, Neomycin, and Chloramphenicol in *Rhizobium japonicum*. Antimicrobial Agents and Chemotherapy. 1973;**4**:248-253

Science. 1982;**215**:1631-1632

[28] Rivas R, Martens M, de Lajudie P, Willems A. Multilocus sequence analysis of the genus *Bradyrhizobium*. Systematic and Applied Microbiology.

[27] Ashraf MA, Maah MJ, Yusoff I. Soil Contamination, Risk Assessment and Remediation. London, UK: IntechOpen;

agee.2017.08.016

2009;**32**:101-110

10.1016/j.agee. 2017.06.017

**170**

1970

[40] Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution. 2016;**33**:1870-1874. DOI: 10.1093/molbev/msw054

[41] Ansari PG, Rao DLN, Pal KK. Diversity and phylogeny of soybean rhizobia in central India. Annales de Microbiologie. 2014;**64**:1553-1565. DOI: 10.1007/s13213-013-0799-2

[42] Appunu C, N'Zoue A, Laguerre G. Genetic diversity of native bradyrhizobia isolated from soybeans (*Glycine max* L.) in different agricultural-ecological-climatic regions of India. Applied and Environmental Microbiology. 2008;**74**:5991-5996. DOI: 10.1128/AEM.01320-08

[43] Guimarães AA, Florentino LA, Almeida KA, Lebbe L, Silva KB, Willems A, et al. High diversity of *bradyrhizobium* strains isolated from several legume species and land uses in Brazilian tropical ecosystems. Systematic and Applied Microbiology. 2015;**38**:433-441. DOI: 10.1016/j. syapm.2015.06.006

[44] Ribeiro PR, Santos JV, Costa EM, Lebbe L, Assis ES, Louzada MO, et al. Symbiotic efficiency and genetic diversity of soybean bradyrhizobia in Brazilian soils. Agriculture, Ecosystems and Environment. 2015;**212**:85-93

[45] Saeki Y, Shiro S, Tajima T, Yamamoto A, Sameshima-Saito R, Sato T, et al. Mathematical ecology analysis of geographical distribution of soybean-nodulating bradyrhizobia in Japan. Microbes and Environments. 2013;**28**:470-478. DOI: 10.1264/jsme2. ME13079

[46] Siqueira AF, Ormeno-Orillo E, Souza RC, Rodrigues EP, Almeida LGP, Barcellos FG, et al. Comparative genomics of *Bradyrhizobium japonicum*, CPAC 15 and *Bradyrhizobium diazoefficiens*, CPAC 7: Elite model strains for understanding symbiotic performance with soybean. BMC Genomics. 2014;**15**:420. DOI: 10.1186/1471-2164-15-420

[47] Soe KM, Bhromsiri A, Karladee D, Yamakawa T. Effects of endophytic actinomycetes and *Bradyrhizobium japonicum* strains on growth, nodulation, nitrogen fixation and seed weight of different soybean varieties. Soil Science and Plant Nutrition. 2012;**58**:319-325. DOI: 10.1080/00380768.2012.682044

[48] Akiyama H, Hoshino YT, Itakura M, Shimomura Y, Wang Y, Yamamoto A, et al. Mitigation of soil N2O emission by inoculation with a mixed culture of indigenous *Bradyrhizobium diazoefficiens*. Scientific Reports. 2016;**6**:1-8. DOI: 10.1038/srep32869

[49] Itakura M, Uchida Y, Akiyama H, Hoshino YT, Shimomura Y, Morimoto S, et al. Mitigation of nitrous oxide emissions from soils by *Bradyrhizobium japonicum* inoculation. Nature: Climate Change. 2013;**3**:208-212. DOI: 10.1038/ nclimate1734

[50] Sameshima-Saito R, Chiba K, Minamisawa K. Correlation of denitirifying capability with the existence of *nap, nir, nor* and *nos* genes in diverse strains of soybean bradyrhizobia. Microbes and Environments. 2006;(3):174-184. DOI: 10.1264/jsme2.21.174

[51] Hayashi M, Saeki Y, Haga M, Harada K, Kouchi H, Umehara Y. *Rj* (*rj*) genes involved in nitrogen-fixing root nodule formation in soybean. Breeding

Science. 2012;**61**:544-553. DOI: 10.1270/ jsbbs.61.544

[52] Devine TE, Kuykendall LD, O'Neill JJ. DNA homology group and the identity of bradyrhizobial strains producing rhizobitoxine-induced foliar chlorosis on soybean. Crop Science. 1988;**28**:939-941. DOI: 10.2135/cropsci 1988.0011183X002800060014x

[53] Eaglesham ARJ, Ayanaba A. Tropical stress ecology of rhizobia, root nodulation and legume nitrogen fixation. In: Subba Rao NS, editor. Selected Topics in Biological Nitrogen Fixation. New Delhi: Oxford/IBH Publishing; 1984. pp. 1-35

**173**

**Chapter 11**

Legumes

*Mohamed Mars*

**Abstract**

**1. Introduction**

Phylogenomic Review of Root

Nitrogen-Fixing Symbiont

Northwestern African Wild

*Mokhtar Rejili, Mohamed Ali BenAbderrahim and* 

**Keywords:** rhizobia, North Africa, symbiosis, legumes, phylogenomic

illustrate the problems and potential for their better exploitation.

Africa has a vast array of indigenous legumes, ranging from large rain forest trees to small annual herbs [1]. However, in recent years, there has been a tendency in agriculture and forestry to use exotic species for crops and wood. As has been pointed out several times over nearly 30 years, most recently [2], by the US National Academy of Sciences, this ignores the potential of the native species, which are arguably better adapted to their environment. For this review, the nodulated indigenous legume genera in Northwestern Africa with known uses have been selected to

The wild legume flora in Northwestern Africa is rich, with great specific and infraspecific diversity [3]. The overgrazing and expansion of agriculture has gradually

The present review discusses the phylogenomic diversity of root nitrogen-fixing bacteria associated to wild legumes under North African soils. The genus *Ensifer* is a dominant rhizobium lineage nodulating the majority of the wild legumes, followed by the genus *Rhizobium* and *Mesorhizobium.* In addition, to the known rhizobial genera, two new *Microvirga* and *Phyllobacterium* genera were described as real nodulating and nitrogen-fixing microsymbiotes from *Lupinus* spp. The promising rhizobia related to nitrogen fixation efficiency in association with some legumes are shared. Phylogenetic studies are contributing greatly to our knowledge of relationships on both sides of the plant-bacteria nodulation symbiosis. Multiple origins of nodulation (perhaps even within the legume family) appear likely. However, all nodulating flowering plants are more closely related than previously suspected, suggesting that the predisposition to nodulate might have arisen only once. The origins of nodulation, and the extent to which developmental programs are conserved in nodules, remain unclear, but an improved understanding of the relationships between nodulin genes is providing some clues.

Population Nodulating

#### **Chapter 11**

*Nitrogen Fixation*

jsbbs.61.544

Science. 2012;**61**:544-553. DOI: 10.1270/

[52] Devine TE, Kuykendall LD, O'Neill JJ. DNA homology group and the identity of bradyrhizobial strains producing rhizobitoxine-induced foliar chlorosis on soybean. Crop Science. 1988;**28**:939-941. DOI: 10.2135/cropsci 1988.0011183X002800060014x

[53] Eaglesham ARJ, Ayanaba A. Tropical stress ecology of rhizobia, root nodulation and legume nitrogen fixation. In: Subba Rao NS, editor. Selected Topics in Biological Nitrogen Fixation. New Delhi: Oxford/IBH

Publishing; 1984. pp. 1-35

**172**
