Multilocus Phylogenetic Characterization of Mesorhizobium-like Nodule-associated Isolates from Root Nodules of Genista acanthoclada

Abstract

The root nodule symbiosis between legumes and rhizobia plays a fundamental role in biological nitrogen fixation and ecosystem sustainability. Genista acanthoclada, a perennial woody legume native to arid and rocky slopes of Western Türkiye, has not previously been investigated regarding to its symbiotic microsymbionts. In this study, five bacterial isolates were obtained from root nodules of G. acanthoclada collected from Yamanlar Mountain (İzmir, Türkiye) on Yeast Extract Mannitol Agar (YEMA) and rhizobial strains (GA.7.1 and GA.9) were rapidly selected by colony PCR targeting the nodC gene. Both isolates exhibited typical rhizobial phenotypic characteristics, including Gram-negative rod-shaped morphology, positive catalase and oxidase activities, and weak Congo red absorption on YEMA medium. Taxonomic analyses based on 16S rRNA, recA, and atpD gene sequences revealed that both isolates belong to the genus Mesorhizobium. The incongruence between the phylogenetic analysis based on nodC amino acid sequences and the core gene phylogenies suggests that horizontal gene transfer may play a role in the evolution of nodulation-related genes. In contrast, the nifH-based phylogeny was largely congruent with housekeeping gene phylogenies, indicating a more conserved evolutionary history of nitrogen fixation genes. These findings provide the first data on the rhizobia associated with the nodules of G. acanthoclada and contribute to a better understanding of the evolutionary dynamics of legume–rhizobium interactions.

Keywords

• Rhizobia
• legume
• symbiotic nitrogen fixation (SNF)
• nodC
• nifH

Supporting Institution

Ege University

Project Number

75599

Ethical Statement

Ethics committee approval is not required for this study.

Thanks

This study was conducted as part of Betül Dağlı’s master’s thesis and was funded by the Ege University Scientific Research Projects Coordination Unit (BAP), Project No. 75599.

Genista acanthoclada'nın Kök Nodüllerinden Elde Edilen Mesorhizobium Benzeri Nodül Ilişkili Izolatların Çoklu Lokus Filogenetik Karakterizasyonu

Abstract

Baklagiller ve Rhizobia arasındaki kök nodül simbiyozu, biyolojik azot fiksasyonunda ve ekosistem sürdürülebilirliğinde temel bir rol oynamaktadır. Batı Türkiye’de kurak ve kayalık yamaçlara özgü çok yıllık odunsu bir baklagil olan Genista acanthoclada, bugüne kadar simbiyotik mikrosimbiyontları açısından incelenmemiştir. Bu çalışmada, Yamanlar Dağı’ndan (İzmir, Türkiye) toplanan G. acanthoclada kök nodüllerinden Yeast Extract Mannitol Agar (YEMA) besiyerinde beş bakteri izolatı elde edilmiş ve nodC genine yönelik koloni PCR analizi ile rhizobiyal suşlar (GA.7.1 ve GA.9) hızlı biçimde seçilmiştir. Her iki izolat, Gram-negatif çubuk şekilli hücre morfolojisi, pozitif katalaz ve oksidaz aktiviteleri ve YEMA ortamında zayıf Kongo kırmızısı emilimi gibi tipik rhizobiyal fenotipik özellikler sergilemiştir. 16S rRNA, recA ve atpD gen dizilerine dayalı taksonomik analizler, her iki izolatın Mesorhizobium cinsine ait olduğunu ortaya koymuştur. nodC amino asit dizilerine dayalı filogenetik analiz ile çekirdek gen filogenileri arasındaki uyumsuzluk, nodülasyonla ilişkili genlerin evriminde yatay gen transferinin rol oynayabileceğini düşündürmektedir. Buna karşılık nifH genine dayalı filogeni, evrimsel kronometre olarak kabul edilen diğer genlerle büyük ölçüde uyumlu bulunmuş ve azot fiksasyon genlerinin daha korunmuş bir evrimsel geçmişe sahip olduğunu göstermiştir. Bu bulgular, G. acanthoclada'nın nodülleriyle ilişkili rhizobia hakkında ilk verileri sağlamakta ve baklagil-rizobiyum etkileşimlerinin evrimsel dinamiklerinin daha iyi anlaşılmasına katkıda bulunmaktadır.

Keywords

• Rhizobia
• baklagil
• simbiyotik azot fiksasyonu (SNF)
• nodC
• nifH

Supporting Institution

Ege Üniversitesi

Project Number

75599

Ethical Statement

Bu çalışma için etik kurul onayı gerekmemektedir.

Thanks

Bu çalışma, Betül Dağlı'nın yüksek lisans tezinin bir parçası olarak yürütülmüş ve Ege Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi (BAP) tarafından 75599 numaralı proje kapsamında finanse edilmiştir.

References

1. Arashida, H., Odake, H., Sugawara, M., Noda, R., Kakizaki, K., Ohkubo, S., ... & Minamisawa, K. (2022). Evolution of rhizobial symbiosis islands through insertion sequence-mediated deletion and duplication. The ISME Journal, 16(1), 112-121. https://doi.org/10.1038/s41396-021-01035-4
2. Boudehouche, W., Parker, M.A., & Boulila, F. (2020). Relationships of Bradyrhizobium strains nodulating three Algerian Genista species. Systematic and Applied Microbiology, 43(3), 126074. https://doi.org/10.1016/j.syapm.2020.126074
3. Chaïch, K., Bekki, A., Bouras, N., Holtz, M.D., Soussou, S., Mauré, L., ... & Cleyet-Marel, J.C. (2017). Rhizobial diversity associated with the spontaneous legume Genista saharae in the northeastern Algerian Sahara. Symbiosis, 71(2), 111-120. https://doi.org/10.1007/s13199-016-0414-y
4. Chun, J., Oren, A., Ventosa, A., Christensen, H., Arahal, D.R., da Costa, M.S., ... & Trujillo, M. E. (2018). Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. International journal of systematic and evolutionary microbiology, 68(1), 461-466.
5. Dinç, M., & Bağcı, Y. (2018). A new species of Genista sect. Spartocarpus (Fabaceae) from Karaman (Turkey). Phytotaxa, 371(1), 49-54. https://doi.org/10.11646/phytotaxa.371.1.6
6. do Vale Barreto Figueiredo, M., do Espírito Santo Mergulhão, A. C., Sobral, J. K., de Andrade Lira Junior, M., & de Araújo, A. S. F. (2013). Biological nitrogen fixation: importance, associated diversity, and estimates. In Plant microbe symbiosis: Fundamentals and advances (pp. 267-289). New Delhi: Springer India. https://doi.org/10.1007/978-81-322-1287-4_10
7. Drew, G.C., Stevens, E.J., & King, K.C. (2021). Microbial evolution and transitions along the parasite–mutualist continuum. Nature Reviews Microbiology, 19(10), 623-638. https://doi.org/10.1038/s41579-021-00550-7
8. Eren Eroglu, A.E., Toklu, K., & Yasa, İ. (2025). Functional genomics of a food-related thermotolerant Acetobacter oryzifermentans strain AAB5: genetic determinants of stress response, CAZyme repertoire, and CRISPR-Cas system. Functional & Integrative Genomics, 25(1), 253. https://doi.org/10.1007/s10142-025-01765-0

9. Eren Eroğlu, A. E., Eroğlu, V., & Yaşa, İ. (2024). Genomic insights into the symbiotic and plant growth-promoting traits of “Candidatus Phyllobacterium onerii” sp. nov. isolated from endemic Astragalus flavescens. Microorganisms, 12(2), 336. https://doi.org/10.3390/microorganisms12020336
10. Ferraz Helene, L.C., Klepa, M.S., & Hungria, M. (2022). New insights into the taxonomy of bacteria in the genomic era and a case study with rhizobia. International journal of microbiology, 2022(1), 4623713. https://doi.org/10.1155/2022/4623713
11. Gaunt, M.W., Turner, S.L., Rigottier-Gois, L., Lloyd-Macgilp, S.A., & Young, J.P. (2001). Phylogenies of atpD and recA support the small subunit rRNA-based classification of rhizobia. International journal of systematic and evolutionary microbiology, 51(6), 2037-2048. https://doi.org/10.1099/00207713-51-6-2037
12. Giraud, E., Moulin, L., Vallenet, D., Barbe, V., Cytryn, E., Avarre, J. C., ... & Sadowsky, M. (2007). Legumes symbioses: absence of Nod genes in photosynthetic bradyrhizobia. Science, 316(5829), 1307-1312. https://doi.org/10.1126/science.1139548
13. Hamza, T. A., & Alebejo, A. L. (2017). Isolation and characterization of rhizobia from rhizospher and root nodule of cowpea, elephant and lab lab plants. The International Journal of Novel Research in Interdisciplinary Studies, 4, 1-7.
14. Hnini, M., & Aurag, J. (2024). Prevalence, diversity and applications potential of nodules endophytic bacteria: a systematic review. Frontiers in Microbiology, 15, 1386742. https://doi.org/10.3389/fmicb.2024.1386742
15. Hungria, M., Campo, R., Chueire, L., Grange, L., & Megias, M. (2001). Symbiotic effectiveness of fast-growing rhizobial strains isolated from soybean nodules in Brazil. Biology and fertility of soils, 33(5), 387-394. https://doi.org/10.1007/s003740100338
16. Janczarek, M., Kozieł, M., Adamczyk, P., Buczek, K., Kalita, M., Gromada, A., ... & Bieganowski, A. (2024). Symbiotic efficiency of Rhizobium leguminosarum sv. trifolii strains originating from the subpolar and temperate climate regions. Scientific Reports, 14(1), 6264. https://doi.org/10.1038/s41598-024-56988-1
17. Jiao, Y S., Yan, H., Ji, Z.J., Liu, Y.H., Sui, X.H., Wang, E.T., ... & Chen, W.F. (2015). Rhizobium sophorae sp. nov. and Rhizobium sophoriradicis sp. nov., nitrogen-fixing rhizobial symbionts of the medicinal legume Sophora flavescens. International journal of systematic and evolutionary microbiology, 65(Pt_2), 497-503. https://doi.org/10.1099/ijs.0.068916-0
18. Kalita, M., & Małek, W. (2020). Root nodules of Genista germanica harbor Bradyrhizobium and Rhizobium bacteria exchanging nodC and nodZ genes. Systematic and Applied Microbiology, 43(1), 126026. https://doi.org/10.1016/j.syapm.2019.126026
19. Kawaka, F. (2022). Characterization of symbiotic and nitrogen fixing bacteria. AMB Express, 12(1), 99. https://doi.org/10.1186/s13568-022-01441-7
20. Kawaka, F., & Muoma, J. (2020). Distribution and phenotypic characteristics of common bean (Phaseolus vulgaris L.) nodulating bacteria in diverse soils. Acta Agriculturae Scandi navica, Section B—Soil & Plant Science, 70(7), 564-571. https://doi.org/10.1080/09064710.2020.1807595
21. Kuzmanović, N., Fagorzi, C., Mengoni, A., Lassalle, F., & Dicenzo, G.C. (2022). Taxonomy of Rhizobiaceae revisited: proposal of a new framework for genus delimitation. International journal of systematic and evolutionary microbiology, 72(3), 005243. https://doi.org/10.1099/ijsem.0.005243
22. Laguerre, G., Nour, S. M., Macheret, V., Sanjuan, J., Drouin, P., & Amarger, N. (2001). Classification of rhizobia based on nodC and nifH gene analysis reveals a close phylogenetic relationship among Phaseolus vulgaris symbionts. Microbiology, 147(4), 981-993. https://doi.org/10.1099/00221287-147-4-981
23. Liu, S., Jiao, J., & Tian, C.F. (2023). Adaptive evolution of rhizobial symbiosis beyond horizontal gene transfer: From genome innovation to regulation reconstruction. Genes, 14(2), 274. https://doi.org/10.3390/genes14020274
24. Lopez, B.D.O., Teixeira, A.F.D.S., Michel, D.C., Guimarães, A.A., Costa, A.M.D., Costa, J.S., ... & Moreira, F.M.D.S. (2022). Genetic and symbiotic characterization of rhizobia nodulating legumes in a mining area in southeast Brazil. Scientia Agricola, 79, e20200238. https://doi.org/10.1590/1678-992X-2020-0238
25. Makkar, K., & Jangra, S. (2017). Isolation and characterization of Rhizobium from Chickpea (Cicer arietinum). International Journal of Current Microbiology and Applied Sciences, 6(11), 2880-2893. https://doi.org/10.20546/ijcmas.2017.611.340
26. Martens, M., Dawyndt, P., Coopman, R., Gillis, M., De Vos, P., & Willems, A. (2008). 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, 58(1), 200-214. https://doi.org/10.1099/ijs.0.65392-0
27. Meier-Kolthoff, J.P., & Göker, M. (2019). TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nature communications, 10(1), 2182. https://doi.org/10.1038/s41467-019-10210-3
28. Ormeno-Orrillo, E., Servín-Garcidueñas, L.E., Rogel, M.A., González, V., Peralta, H., Mora, J., ... & Martínez-Romero, E. (2015). Taxonomy of rhizobia and agrobacteria from the Rhizobiaceae family in light of genomics. Systematic and applied microbiology, 38(4), 287-291. https://doi.org/10.1016/j.syapm.2014.12.002
29. Rajkumari, J., Katiyar, P., Dheeman, S., Pandey, P., & Maheshwari, D.K. (2022). The changing paradigm of rhizobial taxonomy and its systematic growth upto postgenomic technologies. World Journal of Microbiology and Biotechnology, 38(11), 206. https://doi.org/10.1007/s11274-022-03370-w
30. Sarita, S., Sharma, P.K., Priefer, U.B., & Prell, J. (2005). Direct amplification of rhizobial nodC sequences from soil total DNA and comparison to nodC diversity of root nodule isolates. FEMS Microbiology Ecology, 54(1), 1-11. https://doi.org/10.1016/j.femsec.2005.02.015
31. Suzaki, K., Yoshida, K., & Sawada, H. (2004). Detection of tumorigenic Agrobacterium strains from infected apple saplings by colony PCR with improved PCR primers. Journal of General Plant Pathology, 70(6), 342-347. https://doi.org/10.1007/s10327-004-0133-8
32. Tamura, K., Stecher, G., & Kumar, S. (2021). MEGA11: molecular evolutionary genetics analysis version 11. Molecular biology and evolution, 38(7), 3022-3027 https://doi.org/10.1093/molbev/msab120
33. Umesha, S., Chandan, S., & Nanjunda, S. (2012). Colony PCR-single strand confirmation polymorphism for the detection of Ralstonia solanacearum in tomato. International Journal of Integrative Biology, 13(1), 45-51.
34. Vincent, J.M. (1970). A manual for the practical study of the root-nodule bacteria, International Biological Program. IBP Handbook, 1-13.
35. Wang, E.T. (2019). Current systematics of rhizobia. In Ecology and evolution of rhizobia: principles and applications (pp. 41-102). Singapore: Springer Singapore. https://doi.org/10.1007/978-981-32-9555-1_3
36. Yan, J., Han, X.Z., Ji, Z.J., Li, Y., Wang, E.T., Xie, Z.H., & Chen, W.F. (2014). Abundance and diversity of soybean-nodulating rhizobia in black soil are impacted by land use and crop management. Applied and environmental microbiology, 80(17), 5394-5402. https://doi.org/10.1128/AEM.01135-14