The isolates evaluated in this work were obtained by Sampaio FB8 and are available at the Collection of Microorganisms and Multifunctional Fungi of Embrapa Rice and Beans. Strains of Rhizobium tropici (CIAT899 and H12), Rhizobium freirei (PRF81) were used as reference strains in all analyses and, Rhizobium etli bv. phaseoli (CFN42) used in the BOX- and REP-PCR analyses.

CSU was assayed for 98 isolates and for the R. tropici reference strains. Bacteria were kept for growth on modified YMA (Yeast Mannitol Agar) culture medium, without mannitol, added with individual carbon sources sucrose, glucose, malic acid, maleic acid, nicotinic acid, inositol, sorbitol, arabinose, fructose and glycerol. After incubation at 28°C, bacterial growth was verified from 48 to 96h at each 24h.

The same isolates and reference strains were assessed for TST on YMA culture medium on a factorial (5×5) arrangement (concentrations of NaCl – 0%, 1%, 2%, 4%, 6% and temperature – 28°C, 33°C, 38°C, 43°C, 48°C) incubated for a period of 48h.

Based on CSU and TST, 55 isolates were selected for genotypic characterization. Genomic DNA was extracted according to Ausubel et al.9 DNA quantity was estimated by spectrophotometry (NanoDrop®, Thermo scientific, Wilmington, USA), and DNA concentration was adjusted to 50ngμL−1 for all samples. BOX-PCR was performed using the primer BOX A1R (5′-CTACGGCAAGGCGACG-3′), while REP-PCR was performed using the primers REP-1 (5′-IIIICGICGICATCIGGC-3′) and REP-2 (5′-ICGICTTATCIGGCCTAC-3′) according to Versalovic et al.10

The BOX-PCR reaction was performed in a final volume of 15μL, containing 2.9μL of milli-Q water, 7.5μL of 2× QIAGEN Multiplex PCR Master Mix (3mM Mg2+), 1.6μL of primer BOX A1R (50pmolμL−1) and 3μL of DNA template (50ngμL−1). The REP-PCR reaction was performed in a final volume of 15μL, containing 2.0μL of milli-Q water, 7.5μL of 2× QIAGEN Multiplex PCR Master Mix (3mM Mg2+), 1.25μL of each primer REP 1 and REP 2 (10pmolμL−1) and 3μL of DNA template (10ngμL−1).

The amplification program was designed according to Kaschuk et al.11 PCR amplification consisted of an initial denaturing step (95°C; 7min); followed by 35 cycles of denaturation (94°C; 1min), annealing (55°C; 1min for BOX-PCR and 40°C; 1min for REP-PCR) and extension (65°C; 8min); followed by a final extension cycle (65°C; 15min). The PCR program was performed in a thermocycler Biocycler® (Applied Biosystems).

PCR products were subjected to electrophoresis on an agarose gel 1% (50V; 7h) in TAE buffer 0.75×12 using 1kb DNA Ladder® (Norgen) as a DNA band position marker. The agarose gel was stained with SYBR® green (Life Technologies) and visualized with a MultiDoc-it® system.

Based on genotypic characterization, 30 isolates were selected to evaluate their symbiotic efficiency. In addition to the isolates, the treatments were composed of two R. tropici strains (CIAT899 and H12), one R. freirei strain (PRF81), two nitrogen fertilized treatments (NT1=60 and NT2=120kgha−1 of N) and one control treatment (CT – without inoculation and without N).

Seeds of common bean cv. Pérola were sown in autoclaved Leonard jars in a random block design with three replicates. At five days after emergence (DAE), plantlets were inoculated with a cell suspension containing 1×109cellmL−1 of each isolate and reference strain. Once a week, 200mL of nutritive solution without N were added.13 To the nitrogen fertilized treatments NT1 and NT2, 1 and 2mL, respectively, of a solution containing 106.68mgmL−1 of urea were added.

Plants were harvested at 35 DAE. Roots were carefully washed, dried in a paper towel, and the nodules were detached and counted to determine the number of nodules (NN). The leaves were detached from shoots to determine the leaf area (LA) using a leaf area meter LI-COR model 3100. Shoots and nodules were dried (65°C; 72h) to determine the shoot dry mass (SDM) and nodule dry mass (NDM). Subsequently, to determine SDM, shoot of plants were milled to determine the total N (N-Total) using the Kjedahl method, as described by Silva and Queiroz.14

Based on the symbiotic efficiency five isolates (ALSG5A1, JPrG1A1, JPrG6A8, JPrG8A7 and PCG4A2) were selected for 16S rRNA sequencing. The DNA of the isolates was obtained according to Laranjo et al.15 The 16S rRNA region was amplified by PCR reaction using the primers Y1 and Y3.15,16 The amplicons were purified and used on the sequencing reaction according to Laranjo et al.15 Sequences coding for the partial 16S rRNA genes of the isolates ALSG5A1, JPrG1A1, JPrG6A8, JPrG8A7 and PCG4A2 were obtained and when submitted to the GenBank database (www.ncbi.nlm.nih.gov) received the accession numbers KU598665, KU598663, KU598662, KU598664 and KU598661, respectively.

CSU, TST and genotypic characterization data were transformed into a binary matrix. The binary matrices were used for the construction of a similarity matrix using the Jaccard coefficient. The UPGMA (Unweighted pair-group method) was applied to transform the similarity matrix into a similarity dendrogram using NTSYSpc® software.17

Data obtained from the greenhouse experiment were subjected to analysis of variance; when F was significant, the Scott Knott test of means was applied with a 5% probability using SISVAR statistical software.18 Pearson correlation analyses were also performed for NDM and SDM, LA and N-Total using R statistical software.19

For the 16S rRNA-based phylogeny, the sequences obtained were submitted to NCBI BLAST against a non-redundant nucleotide database for getting homologous sequences.20 Sequences showing degree of similarity were aligned using the CLUSTAL W program.21 The evolutionary history was inferred using the Maximum-likelihood method,22 with tree consensus being inferred from 500 replicates using bootstrap.23 The evolutionary distances were computed using the Maximum Composite Likelihood method proposed by Tamura et al.24 All positions containing gaps and missing data were eliminated. There were a total of 989 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.25

CSU evaluation revealed the distribution of 98 isolates among 28 groups, while for TST the isolates were distributed into 27 groups. Among the reference strains, R. tropici CIAT899 grew in all carbon sources, showing the same reference of CSU observed to the 24 isolates (Table S1). Regarding TST, R. freirei PRF81 showed greater tolerance among the reference strains; however, 18 isolates were able to grow under more stringent conditions as compared to R. freirei PRF81 (Table S2).

Similarity analysis based on the CSU and TST data resulted in similarities among isolates varying from 60% to 100%, and five physiological groups (PG) were formed considering a similarity level of 75% (Fig. 1). PG1 comprises the reference strain R. freirei PRF81 and three isolates, representing 3% of the total isolates. The reference strains R. tropici CIAT899 and R. tropici H12 clustered with 49 isolates on PG4, representing 48.5% of the evaluated isolates. Moreover, 38, 7 and 1 isolates formed PG2, PG3 and PG5 clusters, respectively (Fig. 1).

Fig. 1.

Consensus dendrogram obtained by combining CSU and TST data of 98 isolates from wild genotypes of common bean. The dendrogram was generated using the UPGMA algorithm, and the similarity matrix was determined using the Jaccard coefficient.

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Based on the results of physiological clustering (Fig. 1), 55 isolates were selected for genotypic characterization with the REP- and BOX-PCR markers. Using this approach, isolates were distributed among 11 different genotype groups (GG) when considering a 65% similarity (Fig. 2).

Fig. 2.

Consensus dendrogram obtained by combining the REP and BOX-PCR data of 55 isolates from wild genotypes of common bean. The dendrogram was generated using the UPGMA algorithm, and the similarity matrix was determined using the Jaccard coefficient.

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Fingerprinting analysis based on BOX- and REP-PCR markers showed that most of the isolates (65.45%) clustered with the R. tropici reference strains R. tropici CIAT899 and R. tropici H12, forming GG1 with an approximately 65% similarity. The isolates JPrG4A9, UbALG7A7 and PCG4A2 showed an approximately 69% similarity with the reference strain R. freirei PRF81 (GG3). Moreover, the isolate ALSG9A8 showed an approximately 72% similarity with the strain of R. etli bv. phaseoli CFN 42 (GG2). Even with a large fraction of isolates clustering with R. tropici reference strains, no instances of identical fingerprinting were observed among the bacteria studied, indicating high polymorphism among the isolates.

To evaluate symbiotic efficiency in greenhouse conditions, 30 isolates were used based on genotype clustering (Fig. 2). The inoculation significantly affected all of the evaluated parameters (Table 1).

The NN showed significant difference among the isolates. The Significant greater values varied from 133 to 174nodulesplant−1 and, it were found for the isolates ALSG5A4, JPrG6A8, NVSG11A3, UnPaG11A9 and UnPaG8A12. Among the reference strains, the best results were observed for R. tropici CIAT899 and R. tropici H12, with 110 and 96nodulesplant−1, respectively. Those same isolates also showed high NDM values; however, there was no significant difference between the reference strains R. tropici CIAT899 and R. tropici H12. Moreover, 17 isolates showed greater NDM than the R. freirei PRF81 (Table 1).

Regarding SDM, greater values were observed for the treatment corresponding to 120kgha−1 of N (NT2). However, the isolates JPrG6A8, NVSG11A3, NVSG2A2, NVSG7A7, PCG2A5, UbALG3A5, UbALG4A6 and UnPaG8A12 showed values of SDM similar to those obtained by the reference strains R. tropici CIAT899 and R. tropici H12 and by the treatment corresponding to 60kgha−1 of N (NT1) (Table 1).

The effect of the treatments on LA was similar to those on SDM, except for the isolates JPrG6A8, UbALG3A5 and UbALG4A6, which had lower values of LA compared to the reference strains R. tropici CIAT899 and R. tropici H12. Additionally, the isolates NVSG2A6 and UnPaG11A9 showed values equal to those observed for the reference strains R. tropici CIAT899 and R. tropici H12 (Table 1).

Similar to SDM and LA, higher N-Total values were observed for NT1 and NT2 and between inoculated treatments. Among the inoculated treatments, the isolates NVSG2A2 and UnPaG8A12 had N-Total values that were statistically similar to that observed for the reference strain R. tropici CIAT899 (Table 1).

Correlation analysis of NDM with parameters of shoot growth (SDM, N-Total and LA) allowed for the identification of different classes of symbiotic efficiencies, such as few nodulation/few growth, few nodulation/high growth, high nodulation/few growth and high nodulation/high growth. The distribution of the isolates occurred in different quarters of the graphic defined in function of the general mean of the treatments (Fig. 3).

Fig. 3.

Pearson correlation analysis for (NDM) nodule dry mass and (SDM) shoot dry mass. (A) NDM and (N-Total) total nitrogen, (B) NDM and (LA) leaf area, and (C) common bean inoculated with different rhizobia isolates. Dashed lines represent the correlation tendency, and red lines represent the general mean for each parameter. ** p<0.01.

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Correlation analyses between NDM×SDM, NDM×N-Total and NDM×LA showed highly significant results, with correlation coefficients (r) of 0.7, 0.48 and 0.78, respectively. This analysis showed that for the three correlations performed, the isolates UnPaG8A12, NVSG11A3, NVSG2A2, JPrG6A8 and UbALG4A6 and, the reference strain R. tropici CIAT899, were above the general mean (Fig. 3). Remarkably the isolates UbALG3A5 and UnPaG4A9 (Fig. 3A), JPrG1A1, PCG4A6, ALSG7A7 and UnPaG4A9 (Fig. 3B) and UbALG3A5 (Fig. 3C) also showed high values of SDW, N-Total and LA, respectively, even with low values NDM.

Regarding the correlation between NDM and SDM, 10 isolates and the reference strain R. tropici CIAT899 appeared on the upper right quarter, indicating that these bacteria show a high capacity for both nodulation and accumulation of SDM. In contrast, the isolates UbALG3A5 and UnPaG4A9, and the reference strain R. tropici H12, accumulated a high SDM, even with few nodulations (Fig. 3A). The isolates and the reference strain R. freirei PRF81 appearing in the lower left quarter had a symbiotic performance below the general mean, and for those in the lower right quarter, the vegetal growth was weak, even with high nodulation. For the NDM×N-Total correlation, nine isolates and the reference strain R. tropici CIAT899 showed a high symbiotic efficiency, promoting high values for both NDM and N-Total. In addition, the isolates JPrG1A1, PCG4A6, ALSG7A7 and UnPaG4A9 were able to promote high N-Total accumulation with low NDM (Fig. 3B).

Regarding the NDM and LA correlation, twelve isolates and the reference strain R. tropici CIAT899 appeared on the upper right quarter, indicating that these bacteria show high capacity for both nodulation and LA growth, and the isolate UbALG3A5 and the reference strain R. tropici H12 promoted high LA with low NDM (Fig. 3C).

Sequencing analysis revealed the grouping of three isolates (ALSG5A1, JPRG1A1 and JPrG6A8) into the R. tropici and R. freirei cluster, while the other two isolates grouped with Rhizobium leguminosarum bv. viciae, R. leguminosarum bv. phaseoli, Rhizobium fabae, Rhizobium pisi and Rhizobium phaseoli strains (Fig. 4).

Fig. 4.

Maximum-likelihood phylogeny of the 16S rRNA gene showing the relationships between rhizobia isolates obtained from wild genotypes of common bean (in bold) and Rhizobium sp. reference strains. GenBank accession numbers are shown in parentheses. Bar, 2nt substitutions per 1000nt.

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