Definition of the symbiovar viciae in the species Rhizobium azibense and biogeographic implications

Vicia faba L. (faba bean) is a legume cultivated worldwide which commonly establishes effective symbiosis with the symbiovar viciae of species from the Rhizobium leguminosarum phylogenetic group. However, on the basis of the rrs, recA, and atpD gene phylogenies, in this work we identified a strain named EFBRI 42 nodulating V. faba as Rhizobium azibense. This is the first report on the nodulation of Vicia by R. azibense which commonly nodulates P. vulgaris and to date encompasses strains harboring the nodC genes typical of the symbiovars gallicum and phaseoli. However, the strain EFBRI 42 carries a nodC gene typical of the symbiovar viciae for which we report here by the first time this symbiovar in R. azibense. This finding showed the existence of symbiotic genes horizontal transfer events during the coevolution of R. azibense with P. vulgaris and V. faba in their respective distribution centers of Mesoamerica and the Middle East.


Keywords Rhizobium azibense · Symbiovar viciae · Vicia faba · Egypt
Vicia faba L. (faba bean) is a legume probably indigenous to the Near East (Cubero 1974) which is currently cultivated worldwide for the high nutritive value of its seeds, its usefulness as forage and cover crop, and by its ability for nitrogen fixation in symbiosis with rhizobia (Etemadi et al. 2019;Maaluf et al. 2019).
Several of these studies have been carried out in Egypt where this legume has been cultivated for many centuries (Shamseldin et al., 2009;Youseif et al. 2014;Hassan et al. 2015) showing that the strains nodulating V. faba mostly belong to the symbiovar viciae of Rhizobium leguminosarum and R. etli (Shamseldin et al. 2009;Youseif et al. 2014;Hassan et al. 2015); which is a symbiotic variant able to nodulate specifically legumes of the Vicia cross inoculation group (Rogel et al. 2011;Peix et al. 2015). However, some strains effectively nodulating faba bean have not been assigned to a species and symbiovar until now, as occurs with the strain EFBRI 42 isolated in Egypt (Shamseldin et al., 2009). Therefore, in the present study, we identified this strain through the analysis of the core genes, like rrs, recA, and atpD, and the symbiotic gene nodC, which were not previously analyzed for this strain (Shamseldin et al. 2009), and are commonly used for the rhizobia identification at species and symbiovar levels (Peix et al. 2015).
To obtain sequences of these genes, we extracted DNA of the strain EFBRI 42 grown on TY plates (Triptone Yeast Agar) [(Beringer)]  The amplification and sequencing of rrs, recA, atpD and nodC genes were carried out in the conditions and with the primers previously reported (Carro et al. 2012;Gaunt et al. 2001;Laguerre et al. 2001). The obtained sequences were compared with those from GenBank using the BLASTN program (Altschul et al. 1990) and the sequences of the closely related bacteria were downloaded from GenBank for phylogenetic analyses. The sequences were aligned using the Clustal W program (Thompson et al. 1977). The phylogenetic distances were calculated according to Kimura's two-parameter model (Kimura 1980). The phylogenetic trees were inferred using the neighbor joining model (Saitou and Nei 1987) MEGA 7.0 (Kumar et al. 2016) was used for all phylogenetic analyses (Figs. 1 and 2).
The species from the phylogenetic group of R. leguminosarum are the most common endosymbionts of Vicia species and other legumes from its cross inoculation group as Pisum and Lens (Fig. 1). However, the strain EFBRI 42, isolated in Egypt, represented a separate genotype from R. leguminosarum according to the rrs-RFLP pattern analysis (Shamseldin et al. 2009). Accordingly, the sequence of rrs gene of strain EFBRI 42 showed 100% similarity with respect to the species Rhizobium azibense, which encompasses strains nodulating P. vulgaris in different continents and belongs to a group phylogenetically divergent of R. leguminosarum (Fig. 1).
The four species of this phylogenetic group, R. azibense, R. gallicum, R. mongolense, and R. yanglingense, have closely related rrs genes and therefore, the identification of the strain EFBRI 42 was confirmed by the analysis of the recA and atpD housekeeping genes, which allowed the differentiation of Rhizobium species with closely related rrs genes (Peix et al. 2015). The results of the concatenated recA and atpD gene sequences confirmed the affiliation of the strain EFBRI 42 to R. azibense with similarity values higher than 99.0% in both genes (Fig. 2). These results constitute the first report on the nodulation of V. faba by R. azibense, which to date only included strains isolated from P. vulgaris nodules (Mnasri et al. 2014).
The identification at symbiovar level is mainly based on the nodC gene analysis in the case of the genus Rhizobium

Rhizobium azibense 23C2 T (JN624691) Phaseolus vulgaris (Tunisia, Africa)
Neorhizobium galegae LMG 6214 T (X67226) Galega orientalis (Finland, Europe)  (Peix et al. 2015). Based on the results of this analysis the Egyptian strain EFBRI 42 belongs to the symbiovar viciae with its nodC gene being closely related (higher than 98% similarity) to Rhizobium strains isolated from Lens culinaris nodules in Morocco, Syria and Iran (Fig. 3). This is the first report of the symbiovar viciae within the species R. azibense which to date only contains strains nodulating P. vulgaris belonging to the symbiovars gallicum and phaseoli (Fig. 3). The strains of the species R. azibense have been isolated in three different continents, Africa, America and Europe (Mnasri et al. 2014). The strains 23C2 T isolated in Tunisia (Africa) and IE4868 isolated in Mexico (America) belong to the symbiovar gallicum, whereas the strain GR42 isolated in Spain (Europe) belongs to the symbiovar phaseoli (Fig. 3). Both symbiovars, phaseoli and gallicum, have been isolated from P. vulgaris nodules in its distribution centers (Silva et al. 2003;Mnasri et al. 2014;Bustos et al. 2017), which are located in Mesoamerica (Bitocchi et al. 2012). Therefore, probably the symbiotic genes typical of these symbiovars arrived to Europe and Africa together with the seeds of P. vulgaris and were transferred to strains of species indigenous to these continents. This seems to be clear in the case of R. leguminosarum strains carrying the symbiovar phaseoli, because an origin outside America has been proposed for this species (Alvarez-Martínez et al. 2009), but still there are not enough data to hypothesize on the geographical origin of the species R. azibense. Nevertheless, the fact of the existence of the symbiovar viciae within this species opens the door to think that this species has coevolved for long times with Vicia species whose geographical origin has been located in a region of the Middle East that include Egypt (Caracuta et al. 2015). In any case, the existence within R. azibense of three symbiovars to date nodulating legumes indigenous to different continents, as occurs with P. vulgaris and V. faba, proved the existence of horizontal transfer events affecting the symbiotic genes during the coevolution of R. azibense with P. vulgaris and V. faba in their respective distribution centers.

Conclusion
In this study, we report for the first time the nodulation of V. faba by the species R. azibense and the definition of the symbiovar viciae within this species, which to date only contained symbiovars nodulating P. vulgaris, such as gallicum and phaseoli. This finding confirmed the existence of horizontal transfer events affecting the symbiotic genes during the coevolution of R. azibense with different legume hosts. Author contributions AS isolate rhizobial strains, DNA isolation and amplification of 16S rRNA; AP amplify the recA and atpD and nodC genes; AS and AP did the sequences of the genes; EV did the comparisons of the three phylogenetic trees. All the three authors shared the initial writing of the manuscript. EV did revision of the final copy.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).
Data availability Sequences of 16S rRNA, recA, atpD, and nodC genes of strain EFBRI are deposited in the gene bank under accession numbers OP223411, OP381820, OP381822, and OP381821, respectively.

Conflict of interest
The authors declare that there are no conflict of interest.
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

Consent to participate Not applicable.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.