Result of the soil chemical analysis on the 11 locations in the Philippines.
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.
- tropical rhizobia
- genomic diversity
- symbiotic performance
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, 2, 3], alkalinity [3, 4], temperature [1, 5, 6, 7, 8, 9, 10, 11], climate [12, 13], soil water status [14, 15], soil type [2, 14, 16, 17, 18], and soil management or cultural practices [2, 14, 19, 20, 21, 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
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 . 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  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
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.
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  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 . Each nodule was homogenized with sterile distilled water in a microtube and streaked on to a yeast-extract mannitol agar (YMA) plate . 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)  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×
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′ . 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′ . 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.
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
2.5 Single-strain inoculation test
After the amplification and the RFLP treatment of the 16S rRNA gene, a single-strain inoculation test was conducted for all the amplified isolates that shared the same restriction enzymes’ fragment patterns with the USDA
The cultivation of soybean was conducted as described above, but without soil. Each isolate was cultured in a YM broth (YMB)  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−1 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
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 . 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  method was used to construct the phylogenetic trees. The genetic distances were computed using the Kimura 2-parameter model  in the Molecular Evolutionary Genetic Analysis (MEGA v7) software . 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/.
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 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
3.2 Distribution of indigenous soybean bradyrhizobia
As seen in Figure 1, it is evident that the 11 most abundant indigenous soybean rhizobia in the Philippines are classified under the genus
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
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
Presented in Figure 4B is the nodulation test performed on the strains and it can be seen for
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
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
Meanwhile, it was included in a recent report that the distribution and abundance of
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
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
It was expected that the strains which were classified as
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 single-strain inoculation does not seem to influence the amount of N that each strain can fix nor their symbiotic ability.
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
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 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
The authors declare no conflict of interest.
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