Isolation and Characterization of Native Rhizobium Strains Nodulating Some Legumes Species in South Brazzaville in Republic of Congo

Actually, in Republic of Congo, rhizobia have poorly phenotypically and biochemically characterized. This study aimed to characterize native rhizobia. Rhizobia strains were isolated using nodule roots collected on Milletia laurentii, Acacia spp., Albizia lebbeck, and Vigna unguiculata. The strains isolated were characterized microbiologically, biochemically, physiologically, and molecularly identified using 16S rRNA method. The results reported in this study are only for six strains of all 77 isolated: RhA1, RhAc4, RhAc15, RhAc13, RhW1, and RhV3. All native strains were positive to urease activity, negative to cellulase and pectinase activity except for one isolate that showed a positive cellulase activity. Moreover, isolates have grown at 12% of NaCl. On different effects of temperatures, isolates were able to grow up to 44 ̊C and showed good growth at pH from 7 to 9 and the ability to use ten different carbon hydrates sources. The strains were identified as Rhizobium tropici, Rhizobium sp., Mesorhizobium sp. Bradyrhizobium yuanmingense and Bradyrhizobium elkanii. The phylogenetically analysis of the 16S rRNA genes, using a clustering method, allowed us to have a history that is both ancient and stable of four clades among genes with similar patterns. Expanding our awareness of the new legume-rhizobia will be a valuable resource for incorporating an alternative nitrogen fixation approach to consolidate the growth of legumes. These germs can be used in Congolese agriculture to improve yield of crops. How to cite this paper: Teresa, M.S., Goma-Tchimbakala, J., Eckzechel, N.S.A. and Aimé, L.A. (2021) Isolation and Characterization of Native Rhizobium Strains Nodulating Some Legumes Species in South Brazzaville in Republic of Congo. Advances in Bioscience and Biotechnology, 12, 10-30. https://doi.org/10.4236/abb.2021.121002 Received: November 12, 2020 Accepted: January 24, 2021 Published: January 27, 2021 Copyright © 2021 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access


Introduction
Nitrogen is an essential component of tissues and growth of plant. This component can be provided by industrial or biological sources. Nitrogenous fertilizers, the main sources of nitrogen for vegetable growth are industrially produced [1].
On the other hand, plant can get nitrogen through the mineral nutrition or by nitrogen-fixing that occurs across symbiosis between rhizobia and legumes. Cooperation between rhizobia and legumes has already been well studied in the background of a mutual nitrogen-fixing symbiosis [2] [3]. The fixation of symbiotic nitrogen (N 2 ) plays an important role in increasing plant biomass [4].
There is an increasing global need for enhancing food production to meet the needs of the fast-growing human population [5]. The conventional way of increasing agricultural yield through high inputs of chemical nitrogen, phosphate fertilizers and pesticides is not sustainable because of high costs and concerns about global warming, environmental pollution, and safety concerns. In this context, the use of natural soil microorganisms to increase the yield of food crops is an ecological, cost-effective and sustainable alternative tool to the use of chemical fertilizers and pesticides [6]. Several authors claimed that the family of Leguminoseae crops often associated with root nodulating bacteria improves nutritional stress by fixing atmospheric nitrogen [7] [8]. At the same time, legumes crops become able to access atmospheric di-nitrogen (N 2 ) by the symbiotic relationship with rhizobia that domicile within root nodules [9]. These symbioses are important interactions on the ecological level, influencing composition, diversity and succession of communities. They contribute to approximately 100 to 290 million tons of N per year to natural ecosystems and improving the growth of fodder plants and cultivated plants of agronomic importance worldwide. Also, bacteria have led to increased diversification of legumes thanks to nitrogen fixation [8]. The particular meaningful of plant-biotic reciprocal action is the mutualism between legume-rhizobia, within rhizobia fix atmospheric nitrogen to get carbon back supplied by the legume hosts, this mutualism adjusts to ensure accuracy regularly the nutrient cycles in natural ecosystems by making nitrogen available in agricultural environments [10]. Rising costs and the negative effects of pesticides and fertilizers on agricultural production necessitate addressing this problem through the use of organic products and natural crop protection options. The manipulation of plant growth-promoting (PGP) bacteria, for better plant health and betterment of soil, has become one of the captivating approaches for developing sustainable farming systems due to their particular respect for the environment, their production, and low cost and reduced con-  [11].
In Africa, a lot of studies showed that Legume-rhizobium symbiosis is one of the most successful models of co-evolution mutualisms between prokaryote and Eukaryote [12]- [17].

Plant Collection
Two sites were chosen for sampling of leguminous. The first site was situated at the Service National de Reboisement (SNR) of Brazzaville and the second site was at Mfilou in a farmer's field. Table 1

Nodules Sampling and Rhizobia Isolation
Nodules were carefully detached from the root system plant and washed several

Biochemical Characterization
Rhizobial strains obtained from all locations were purified and characterized.
The characterization included Gram test with KOH, catalase and oxidase test, macroscopic and microscopic observations. The growth on YMA containing bromothymol blue (YMA + BTB) was also observed.

Rhizobium/Agrobacterium Differentiation Tests
For this experiment, strains were grown on glucose-peptone agar (GPA) medium for 3 days at 28˚C, they were also tested for 3-ketolactose following recommendations of [26]. Rhizobia strains are not able to grow or have a poor growth on GPA and are negative for 3-ketolactose test. Only strains which were both negative for growth on GPA and 3-ketolactose test were selected for the following analysis.  log OD log OD − is the difference between two values of log 10 of OD 2 at T 2 and OD 1 at T 1 [27].

Urease, Cellulase and Pectinase Activity
All native strains after being identified with classical methods were tested for urease, cellulase and pectinase activity [28]. For urease activity, strains were grown on YMA medium supplemented with 2% of urea and 0.012 g of phenol

Use of Carbon Hydrates
Mannitol, Glucose, Maltose, Fructose, Saccharose, Galactose, Lactose, Sorbitol, and Xylose were tested as carbon sources in the growth of strains. In YM medium, Mannitol as carbon sources was substituted by 1% of one of carbon hydrates cited above. Strains were incubated at 30˚C for 3 days. At the end of incubation an Optical Density were taken at 600 nm.

Heavy Metals and Antibiotics Resistance
The heavy metal resistance was tested by using wells method on trypton yeast extract (TY) solid medium containing in g/L: trypton 5 g, yeast extract 3 g, CaCl 2 0.87 g, agar 15 g. Petrie dishes were incubated at 30˚C for 24 to 48 hours. Mercury (Hg), lead (Pb), copper (Cu), silver (Ag), cobalt (Co), zinc (Zn), and nickel (Ni) with concentrations from 10 to 5000 µg/mL were tested. The diameter of inhibition zone (clear zone) was measured in centimeter (cm) after 3 days of incubation, strains were considered as resistant when no inhibition were observed around wells. Antibiogramme and his interpretation were done using the European Committee recommendations on Antimicrobial Susceptibility Testing (EUCAST, 2019).

Sequencing and Molecular Identification
The primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3'), 1492R Alignment of sequences was done with CHROMAS Pro to create the complete counting of 16S rRNA gene. Sequences were blasted on the NCBI GenBank database (https://www.ncbi.com/) for identification of strains. They were also submitted on NCBI GenBank database in order to obtain accession numbers.

Phylogenetic Analysis of 16S Nucleotides
The 16S rRNA nucleotides sequences with similarity ≥ 99% to the reference bacteria (

Isolation of Native Rhizobia from Legumes Species
Atotal of 77 strains were obtained and divided as follows: 25 from Acacia spp, 15 from Millettia laurentii, 17 from Alibizia lebeck and 20 from Vigna unguiculata. Strains from Acacia spp. and Albizia lebeck appeared on the culture medium after 5 days whereas Vigna unguiculata strains grown on the medium appeared after 8 days and those of Millettia laurentii after 9 days. They were white or pink on YMA+CR, translucid or opaque. The strains were Gram-negative, Catalase and Oxidase positives. Microscopic observations revealed that strains were bacteria rod-shapped and motile. All Vigna unguiculata and Milletia laurentii strains were slow-growing bacteria. They took 7 to 9 days to appear on the YMA medium after isolation while Albizia lebeck strains were fast-growing bacteria.
These last strains appeared on the medium 3 days after isolation. The result founded in this study showed that there were among Acacia spp strains both fast and slow-growing strains, they took 3 to 5 days to appear on the YMA medium.
All the fast-growing strains have changed the color of YMA+BTB medium from green to yellow while slow-growing strains from green to blue. Among the 77 strains obtained, 30 were not able to grow on GPA+VB while 12 were negative to 3 keto-lactose tests (Figure 1(a)). Only 6 strains were both unable to grow on GPA and were 3 ketolactose negative. These strains were selected for further analysis. The growth curve (Figure 1(b)) of strains showed for most of them that the exponential phase begins after 2 days and the stationary phase after seven days. All strains in this study were Gram-negative (−), Catalase positive (+), and Oxydase positive (+).

Secondary Metabolites Production and Beneficial Traits
To examine the secondary metabolism of strains, urease, pectinase and cellulase activities were tested. All the tested isolates were urease positive (Figure 2

Physiological Characteristic of Strains
The strains have grown on YM medium with different pH. The growth observed was higher on alkaline medium than on acidic medium. The best growth was observed with RhAc13 on neutral pH (Figure 3(a)). Strains growth was better at   (Figure 3(b)). The results showed that the growth of RhV3 was not affected by the increase of temperature. The growth of strains decreased as the amount of NaCl is increased (Figure 3(c)).
The best growth was observed at 0% of NaCl, all native strains were able to grow until 12% of NaCl. However, the growth was weak at 12% of NaCl in the medium. NaCl seems to have no effect on the growth of RhV3 strain from Vigna unguiculata root nodules because the growth was similar in the range of concentrations used.

Carbon Sources Use by Rhizobium Strains
To detect whether variating carbon sources associated with the growth of strains, ten of them such as mannitol, maltose, arabinose, lactose, galactose, xylose, fructose, saccharose, sorbitol and glucose were tested. The results showed that, all strains were able to use all the carbohydrate tested ( Figure 4). RhAc13 showed the greatest activity on the set of carbon sources except with xylose and fructose. The second-best growth was observed with RhA1. For this strain the smallest growth occurs in presence of Arabinose, whereas RhAc4 showed the lowest growth with Sorbitol and Mannitol. In other hand, the weakest growth of RhW1 was noted with maltose and fructose as carbon source. Figure 4 revealed that the lowest growth of RhAc15 was observed in the presence of arabinose.

Heavymetals Effectson Native Rhizobium Strains
The effects of heavy metals were shown in Figure 5(a) and Figure 5(b). This figure showed that all the strains were resistant to Ag, Pb and Co at 2500 µg/mL ( Figure 5(a)). About 50% of strains tolerated Hg at 10 µg/mL, 33.33% at 50 µg/mL and 16.66% at 100 µg/mL ( Figure 5(b)). About 50% of strains resisted to CuO 2 at 5000 µg/mL and the other 50% resisted at 2500 µg/mL. 83.33% of strains resisted to Ni at 2500 µg/mL while 16.66% resisted at 1000 µg/ml. 66.66% of strains resisted to Zn at 5000 µg/ml and 33.33% at 2500 µg/ml (Figure 5(a) and    (Figure 6(d), Figure 6(f), Figure   6(g), and Figure 6(j)). RhAc15 resisted to all antibiotic except Chloramphenicol for which it was intermediate ( Figure 6(d), Figure 6(f), Figure 6(g), Figure 6(h), and Figure 6(j)). The result showed that RhAc4 resisted to all the tested  Figure 6(g), Figure 6(h), and Figure 6(j)). In other hand the strains RhW1 and RhV3 isolated respectively from Milletia laurentii and Vigna unguiculata were sensitive to all antibiotics except rifampicin for which they were intermediate ( Figure 6). RhA1 resisted to Oxacillin ( Figure   6(j)). Albizia lebeck strain RhA1 was resistant to Erythromycin, Chloramphenicol, Kanamycin and Rifampicin. (Figure 6(b), Figure 6(c), Figure 6(e), Figure   6(i)) but it was sensitive to all others. gel of purified genomic DNA extracted from our bacteria strains. The amplification by PCR of 16S rRNA gene using the genomic DNA, revealed by automated capillary electrophoresis (Qiaxcel) showed bands around 1500 bp (Figure 7(b)). The Blastn of all 16S genes sequences revealed that strains were related with nitrogen-fixing bacteria, belonging to genera such as Rhizobium, Mesorhzobium, and Bradyrhizobium.

Phylogenetic Tree of the Strains
The phylogenetic analysis of the nucleotide's sequences related to 16S rRNA using MEGA X software showed a clear diversification of native strains nodulating legumes speciesin the soil of south of Brazzaville (Figure 8(a)). The phylogenetic tree showed that the strains were grouped into four distinct clades classified I to IV (Figure 8(b)). Clade I comprised test strains that clustered with lineages of Rhizobium tropici and Agrobacterium radiobacter in the 16S housekeeping gene phylogenies. However, All the strains in Clade II, III, and IV formed a monophyletic group, with very close relatedness to any known type strains for example Mesorhizobium loti MAFF 303099, Bradyrhizobium yuanmingense strain B071 16S.

Discussion
In this study, strain tested for growth promotional efficacy under different conditions was focused on a native rhizospheric bacterial isolate of legumes species.
Microbiota should provide native microorganisms with competitive advantages compared to exotic species with the co-existence for many years with the natural soil [31]. We obtained strains that were white or pink on YEMA RC, translucid or opaque Gram-negative, catalase-positive, and oxidase-positive. The strains were both slow-growing bacteria turning YMA+BTB in bleu and fast-growing bacteria turning YMA+BTB in yellow. Similar characteristics have been demonstrated classifying rhizobia in two groups regarding the apparition time during incubation: fast-growing bacteria for those which appear 3 days maximum after incubation and the slow-growing bacteria which appear after 3 days of incubation [25]. Regarding appearance after incubation, our strains were both slow and fast-growing bacteria; but concerning generation time they were all considerate as slow-growing bacteria.
Rhizobia can produce some enzymes such as urease, amylase, cellulase, pectinase [28]. In the present study, all native strains showed a positive urease activity, negative to cellulase, and pectinase activity except for one strain which shown a positive cellulase activity. Our findings were similar to strains isolated from the root nodule of Dalbergia sissoo concerning urease activity [32]. However, strains associated with root nodule of Hedysarum generashown a negative cellulase activity [33] while a positive cellulase activity with rhizobia isolated from Genista cinerea root nodules [28]. Concerning pectinase activity, our results differed with strains that showed positive pectinase activity [28].  [38]. Our results are also close to those already reported on slow-growing rhizobia at a temperature above 40˚C [43].
Slow-growing bacteria are more tolerant to low pH than fast-growing strains [44], confirmed by Bradyrhizobium and Burkholderia strains that are able to grow at pH3 [45]. In this study, all the strains had good growth at basic pH between 7 and 9, and the similar results were obtained with chickpea rhizobia, strains were able to grow at pH between 5 and 9.5 [38]. In the same way, strains growth at pH between 5 and 8 respectively from nodule were confirmed [32] [37]. However, rhizobia of Vigna unguiculata L. Walp strains grow at pH 10 (250 µg/ml), but they are insensitive to HgCl 2 (10 µg/ml) and CuCl 2 (50 µg/ml) [38]. However, rhizobia strains isolated from Vigna Unguiculata resisted to HgCl 2 , CuCl 2 , ZnCl 2 and Pb(CH 3 COO) 2 at low concentrations (100 -200 μg/mL) [46]. The results founded in this study are similar to those obtained by [38] only for mercury [38] and totally similar to those obtained by [46]. Tolerance in abiotic factors, resistance to antibiotics, and heavy metals are important features in the selection of strains for biofertilizers [54] [55].
The sequenced 16S RNA gene showed that all the fast-growing strains belonged to Rhizobium genera while the slow-growing were assigned to Mesorhizo-  [58]. These reports were similar to our finding in the fact that we found Alibizia lebeck nodulated by Rhizobium tropici, Acacia by Rhizobium, and Mesorhizobium. Analyzing the 16S rRNA genes phylogenetically, using a clustering method allowed us to identify four clades among genes with similar patterns, which often indicates that they are active in the same biological processes. The molecular analysis results were consistent with the physiological, morphological, and biochemical traits of the native strains.

Conclusion
In the present study, 77 isolates were obtained from root nodules of four legu- Among these isolates six were picked after Rhizobium-Agrobacterium tests.
Them, they were submitted to different biochemical and molecular tests. The main conclusion was that the bacterial community that nodulating the leguminous was greatly diversified. Thereby, the same plant can be nodulated by several kinds of germs. For example, Milletia laurentii was nodulated by a single species Bradyrhizobium elkanii, while the Accacia spp. could be nodulated by Rhizobium tropici, Rhizobium sp. and Mesorhizobium sp. Albizia lebbeck was nodulated by Rhizobium tropici and Vigna unguiculata by Bradyrhizobium yuanmingense. These species could be used to constitute a bank of bacteria nodulating leguiminous. These bacteria could be used in the Congolese agrosystems to improve yield of crops.