This study identified the phenotypic and genotypic characteristics of the bacteria that nodulate wild Lathyrus and Vicia species natural distribution in the Gaziantep province of Turkey. Principle component analysis of phenotypic features revealed that rhizobial isolates were highly resistant to stress factors such as high salt, pH and temperature. They were found to be highly sensitive to the concentrations (mg/mL) of the antibiotics neomycin 10, kanamycin, and tetracycline 5, as well as the heavy metals Ni 10, and Cu 10, and 5. As a result of REP-PCR analysis, it was determined that the rhizobial isolates were quite diverse, and 5 main groups and many subgroups being found. All of the isolates nodulating wild Vicia species were found to be related to Rhizobium sp., and these isolates were found to be in Clades II, III, IV, and V of the phylogenetic tree based on 16S rRNA. The isolates that nodulated wild Lathyrus species were in Clades I, II, IV, V, VI, VII, and VIII, and they were closely related to Rhizobium leguminasorum, Rhizobium sp., Phyllobacterium sp., Serratia sp., and Pseudomonas sp. According to the genetic analyses, the isolates could not be classified at the species level, the similarity ratio was low, they formed a distinct group that was supported by strong bootstrap values in the phylogenetic tree, and the differences discovered in the network analysis revealed the diversity among the isolates and gave important findings that these isolates may be new species.

Biology Department Science and Letter Faculty Gaziantep University Gaziantep Turkey

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Adiguzel A, Ogutcu H, Baris O, Karadayi M, Gulluce M, Sahin F (2010) Isolation and characterization of rhizobium strains from wild vetch collected from high altitudes in Erzurum-Turkey. Rom Biotechnol Lett 15:5017–5024

Ahmad MH, Uddin MR, McLaughlin W (1984) Characterization of indigenous rhizobia from wild legumes. FEMS Microbiol Lett 24:197–203 DOI

Alexandre A, Laranjo M, Oliveira S (2006) Natural populations of chickpea rhizobia evaluated by antibiotic resistance profiles and molecular methods. Microb Ecol 51:128–136 PubMed DOI

Amarger N, Macheret V, Laguerre G (1997) Rhizobium gallicum sp. nov. and Rhizobium giardinii sp. nov., from Phaseolus vulgaris nodules. Int J Syst Bacteriol 47:996–1006 PubMed DOI

Ampomah OY, Huss-Danell K (2016) Genetic diversity of rhizobia nodulating native Vicia spp. in Sweden. Syst Appl Microbiol 39:203–210 PubMed DOI

Amsalu A, Assefa F, Hailemariam A (2012) Symbiotic and phenotypic characterization of rhizobium isolates of field pea (Pisum sativum L.) fabecae, from central and southern Ethiopia. Ethiop J Biol Sci 11:163–179

Aoki S, Kondo T, Prévost D, Nakata S, Kajita T, Ito M (2010) Genotypic and phenotypic diversity of rhizobia isolated from Lathyrus japonicus indigenous to Japan. Syst Appl Microbiol 33:383–397 PubMed DOI

Basbuga S, Basbuga S, Yayla F, Mahmoud AM, Can C (2021) Diversity of rhizobial and non-rhizobial bacteria nodulating wild ancestors of grain legume crop plants. Int Microbiol 24:207–218 PubMed DOI

Bernal G, Graham PH (2001) Diversity in the rhizobia associated with Phaseolus vulgaris L. in Ecuador, and comparisons with Mexican bean rhizobia. Can J Microbiol 47:526–534 PubMed DOI

Berrada H, Nouioui I, Houssaını MI, Gtarı M, Benbrahım KF (2012) Phenotypic and genotypic characterizations of rhizobia isolated from root nodules of multiple legume species native of Fez, Morocco. African J Microbiol Res 6:5314–5324

Broughton WJ, Dilworth MJ (1970) Control of leghaemoglobin synthesis in snake beans. J Biochem 125:1075–1080. https://doi.org/10.1042/bj1251075 DOI

Bryant D, Moulton V (2004) Neighbor-Net: an agglomerative method for the construction of phylogenetic networks. Mol Biol Evol 21:255–265 PubMed DOI

da Silva VB, da Silva AF, da Silva TR, dos Santos JWM, da Silva JF, de Souza AP, de Freitas ADS, Fernandes-Júnior PI (2019) Fast and efficient symbiotic gene-based duplex PCR approach for the preliminary selection of legume root nodule bacteria. Rhizosphere 10:100144. https://doi.org/10.1016/j.rhisph.2019.100144 DOI

Davis PH (1970) Flora of Turkey and the East Aegean Islands, vol 3. University Press, Edinburgh

de Bruijn FJ (1992) Use of repetitive (repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus) sequences and the polymerase chain reaction to fingerprint the genomes of Rhizobium meliloti isolates and other soil bacteria. Appl Environ Microbiol 58:2180–2187 PubMed DOI PMC

De Meyer SE, Van Hoorde K, Vekeman B, Braeckman T, Willems A (2011) Genetic diversity of rhizobia associated with indigenous legumes in different regions of Flanders (Belgium). Soil Biol Biochem 43:2384–2396. https://doi.org/10.1016/j.soilbio.2011.08.005 DOI

Doyle JJ (1994) Phylogeny of the legume famıly: an approach to understanding the origins of nodulation. Annu Rev Ecol Syst 25:325–349 DOI

Drouin P, Prévost D, Antoun H (1996) Classification of bacteria nodulating Lathyrus japonicus and Lathyrus pratensis in northern Quebec as strains of Rhizobium leguminosarum biovar viciae. Int J Syst Bacteriol 46:1016–1024 PubMed DOI

El-Batanony NH, Castellano-Hinojosa A, Correa-Galeote D, Bedmar EJ (2020) Phylogenetic diversity of bacterial strains from root nodules of legumes grown wild in Egypt. Biocatal Agric Biotechnol 27:101692 DOI

El-Hilali I (2006) Rhizobium-Lupine symbiosis: micro-symbioses biodiversity and highlighting of a multi nodular infection in Lupinus luteus. Dissertation, Dr Univ Mohammed V Agdal, Rabat

Gao J-L, Turner SL, Kan FL, Wang ET, Tan ZY, Qiu YH, Gu J, Terefework Z, Young JPW, Lindström K et al (2004) Mesorhizobium septentrionale sp. nov. and Mesorhizobium temperatum sp. nov., isolated from Astragalus adsurgens growing in the northern regions of China. Int J Syst Evol Microbiol 54:2003–2012. https://doi.org/10.1099/ijs.0.02840-0 PubMed DOI

Germano MG, Menna P, Mostasso FL, Hungria M (2006) RFLP analysis of the rRNA operon of a Brazilian collection of bradyrhizobial strains from 33 legume species. Int J Syst Evol Microbiol 56:217–229. https://doi.org/10.1099/ijs.0.02917-0 PubMed DOI

Gök M, Martin P (1993) Farklı Rhizobium bakterileri ile aşılamanın soya, üçgül ve fiğde simbiyotik azot fiksasyonuna etkisi. Doğa-Tr J Agric for 17:753–761

Graham PH, Draeger KJ, Ferrey ML, Conroy MJ, Hammer BE, Martinez E, Aarons SR, Quinto C (1994) Acid pH tolerance in strains of Rhizobium and Bradyrhizobium, and initial studies on the basis for acid tolerance of Rhizobium tropici UMR1899. Can J Microbiol 40:198–207 DOI

Gritli T, Ellouze W, Chihaoui SA, Barhoumi F, Mhamdi R, Mnasri B (2020) Genotypic and symbiotic diversity of native rhizobia nodulating red pea (Lathyrus cicera L) in Tunisia. Syst Appl Microbiol. https://doi.org/10.1016/j.syapm.2019.126049 PubMed DOI

Hamdi Y. (1997) Symbiotic biological nitrogen fixation proceedings of the training course on bio-organic farming systems for sustainable agriculture. In: Cairo- Egypt. p. 79–102

Han TX, Wang ET, Wu LJ, Chen WF, Gu JG, Gu CT, Tian CF, Chen WX (2008) Rhizobium multihospitium sp. nov., isolated from multiple legume species native of Xinjiang. China Int J Syst Evol Microbiol 58:1693–1699. https://doi.org/10.1099/ijs.0.65568-0 PubMed DOI

Han TX, Tian CF, Wang ET, Chen WX (2010) Associations among rhizobial chromosomal background, nod genes, and host plants based on the analysis of symbiosis of indigenous rhizobia and wild legumes native to Xinjiang. Microb Ecol 59:311–323. https://doi.org/10.1007/s00248-009-9577-x PubMed DOI

Haukka K, Lindström K, Young JPW (1998) Three phylogenetic groups of nodA and nifH genes in Sinorhizobium and Mesorhizobium isolates from leguminous trees growing in Africa and Latin America. Appl Environ Microbiol 64:419–426 PubMed DOI PMC

Huson DH, Bryant D (2005) Application of phylogenetic networks in evolutionary studies. Mol Biol Evol 23:254–267. https://doi.org/10.1093/molbev/msj030 PubMed DOI

Kalkancı N, Şimşek T (2020) Efficiency status of Gaziantep soils. Pist Res J 8:8–11

Kan FL, Chen ZY, Wang ET, Tian CF, Sui XH, Chen WX (2007) Characterization of symbiotic and endophytic bacteria isolated from root nodules of herbaceous legumes grown in Qinghai-Tibet plateau and in other zones of China. Arch Microbiol 188:103–115. https://doi.org/10.1007/s00203-007-0211-3 PubMed DOI

Kantar F (1997) Proceeding of training course on bio-organic farming systems for sustainable agriculture. In: Cairo- Egypt. p. 270–272

Karaduman A, Çimrin K (2016) Nutrient status of Gaziantep agricultural soils and their relationships with some soil properties. KSU J Agric Nat 19:117–129

Kawaka F, Makonde H, Dida M, Opala P, Ombori O, Maingi J, Muoma J (2018) Genetic diversity of symbiotic bacteria nodulating common bean (Phaseolus vulgaris) in western Kenya. PLoS ONE 13:e0207403 PubMed DOI PMC

Kim DH, Kaashyap M, Rathore A, Das RR, Parupalli S, Upadhyaya HD, Gopalakrishnan S, Gaur PM, Singh S, Kaur J et al (2014) Phylogenetic diversity of Mesorhizobium in chickpea. J Biosci 39:513–517. https://doi.org/10.1007/s12038-014-9429-9 PubMed DOI

Küçük Ç, Kivanc M (2008) Preliminary characterization of Rhizobium strains isolated from chickpea nodules. African J Biotechnol 7:772–775

Küçük Ç, Kivanç M, Kinaci E (2006) Characterization of Rhizobium sp. isolated from bean. Turkish J Biol 30:127–132

Laguerre G, Mavingui P, Allard MR, Charnay MP, Louvrier P, Mazurier SI, Rigottier-Gois L, Amarger N (1996) Typing of rhizobia by PCR DNA fingerprinting and PCR-restriction fragment length polymorphism analysis of chromosomal and symbiotic gene regions: application to Rhizobium leguminosarum and its different biovars. Appl Environ Microbiol 62:2029–2036 PubMed DOI PMC

Laguerre G, Nour SM, 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 147:981–993. https://doi.org/10.1099/00221287-147-4-981 PubMed DOI

Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. John Wiley & Sons, Ltd., New York, pp 115–175

Lebrazi S, Chraibi M, Fadil M, Barkai H, Fikri-Benbrahim K (2018) Phenotypic, genotypic and symbiotic characterization of rhizobial isolates nodulating Acacia sp. in Morocco. J Pure Appl Microbiol 12:249–263 DOI

Legesse S, Assefa F (2014) Symbiotic and phenotypic characteristics of rhizobia nodulating Faba bean (Vicia faba) From Tahtay Koraro, Northwestern Zone Of Tigray Regional State, Ethiopia. Int J Technol Enhanc Emerg Eng Res 2:15–23

Lei X, Wang ET, Chen WF, Sui XH, Chen WX (2008) Diverse bacteria isolated from root nodules of wild Vicia species grown in temperate region of China. Arch Microbiol 190:657–671. https://doi.org/10.1007/s00203-008-0418-y PubMed DOI

Li Y, Wang ET, Liu Y, Li X, Yu B, Ren C, Liu W, Yunzhao Li, Xie Z (2016) Rhizobium anhuiense as the predominant microsymbionts of Lathyrus maritimus along the Shandong Peninsula seashore line. Syst Appl Microbiol 39:384–390 PubMed DOI

Mahmoud MA, Mart D, Can C (2019) Phenotypic characterization of indigenous rhizobia nodulating chickpea in Turkey reveals high diversity. Legum Res 42:379–384. https://doi.org/10.18805/LR-430 DOI

Mantelin S, Saux MF-L, Zakhia F, Béna G, Bonneau S, Jeder H, de Lajudie PM, Cleyer Marel JC (2006) Emended description of the genus Phyllobacterium and description of four novel species associated with plant roots: Phyllobacterium bourgognense sp. nov., Phyllobacterium ifriqiyense sp. nov., Phyllobacterium leguminum sp. nov. and Phyllobacterium brassic. Int J Syst Evol Microbiol 4:827–839 DOI

Martínez-Romero E, Caballero-Mellado J (1996) Rhizobium phylogenies and bacterial genetic diversity. CRC Crit Rev Plant Sci 15:113–140. https://doi.org/10.1080/07352689609701938 DOI

Missbah El Idrissi M, Lamin H, Bouhnik O, Lamrabet M, Alami S, Jabrone Y, Bennis M, Bedmar EJ, Abdelmoumen H (2020) Characterization of Pisum sativum and Vicia faba microsymbionts in Morocco and definition of symbiovar viciae in Rhizobium acidisoli. Syst Appl Microbiol 43:126084. https://doi.org/10.1016/j.syapm.2020.126084 PubMed DOI

Mohammed MA, Chernet MT, Tuji FA (2020) Phenotypic, stress tolerance, and plant growth promoting characteristics of rhizobial isolates of grass pea. Int Microbiol 23:607–618. https://doi.org/10.1007/s10123-020-00131-3 PubMed DOI

Moschetti G, Peluso AL, Protopapa A, Anastasio M, Pepe O, Defez R (2005) Use of nodulation pattern, stress tolerance, nodC gene amplification, RAPD-PCR and RFLP-16S rDNA analysis to discriminate genotypes of Rhizobium leguminosarum biovar viciae. Syst Appl Microbiol 28:619–631. https://doi.org/10.1016/j.syapm.2005.03.009 PubMed DOI

Mutch L, Young JPW (2004) Diversity and specificity of Rhizobium leguminosarum biovar viciae on wild and cultivated legumes. Mol Ecol 13:2435–2444. https://doi.org/10.1111/j.1365-294X.2004.02259.x PubMed DOI

Nushair AM, Saha AK, Rahman MA, Mohanta MK, Haque MF (2017) Characterization of indigenous Rhizobium strain isolated from lentil in Rajshahi, Bangladesh. Asian J Adv Basic Sci 5:21–25

Rai R, Dash PK, Mohapatra T, Singh A (2012) Phenotypic and molecular characterization of indigenous rhizobia nodulating chickpea in India. J Exp Biol 50:340–350

Ruiz-Díez B, Fajardo S, del Rosario de Felipe M, Fernández-Pascual M, (2012) Characterization of rhizobia from legumes of agronomic interest grown in semi-arid areas of Central Spain relates genetic differences to soil properties. J Basic Microbiol 52:66–78. https://doi.org/10.1002/jobm.201100058 PubMed DOI

Saïdi S, Ramírez-Bahena MH, Santillana N, Zúñiga D, Álvarez-Martínez E, Peix A, Mhamdi R, Velázquez E (2014) Rhizobium laguerreae sp. nov. nodulates Vicia faba on several continents. Int J Syst Evol Microbiol 64:242–247. https://doi.org/10.1099/ijs.0.052191-0 PubMed DOI

Santillana N, Ramírez-Bahena MH, García-Fraile P, Velázquez E, Zúñiga D (2008) Phylogenetic diversity based on rrs, atpD, recA genes and 16S–23S intergenic sequence analyses of rhizobial strains isolated from Vicia faba and Pisum sativum in Peru. Arch Microbiol 189:239–247. https://doi.org/10.1007/s00203-007-0313-y PubMed DOI

Shiraishi A, Matsushita N, Hougetsu T (2010) Nodulation in black locust by the Gammaproteobacteria Pseudomonas sp. and the Betaproteobacteria Burkholderia sp. Syst Appl Microbiol 33:269–274. https://doi.org/10.1016/j.syapm.2010.04.005.h PubMed DOI

Somasegaran P, Hoben HJ (1994) Handbook for rhizobia: methods in legume-rhizobium technology. 1st ed. Springer Verlag New York, Inc

Sui X, LiLi H, EnTao W, Feng J, YiHai L, WenXin C (2009) Novel associations between rhizobial populations and legume species within the genera Lathyrus and Oxytropis grown in the temperate region of China. Sci China Ser C Life Sci 52:182–192. https://doi.org/10.1007/s11427-008-0132-7 DOI

Tamura K, Stecher G, Kumar S (2021) MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol 38:3022–3027. https://doi.org/10.1093/molbev/msab120 PubMed DOI PMC

Tan A (1996) Turkey: country report to the FAO International Technical Conference on Plant Genetic Resource, Leipzig, Germany

Tanksley SD, McCouch SR (1997) Seed banks and molecular maps: unlocking genetic potential from the wild. Science 277:1063–1066. https://doi.org/10.1126/science.277.5329.1063 PubMed DOI

Tena W, Wolde-Meskel E, Degefu T, Walley F (2017) Genetic and phenotypic diversity of rhizobia nodulating chickpea (Cicer arietinum L.) in soils from southern and central Ethiopia. Can J Microbiol 63:690–707. https://doi.org/10.1139/cjm-2016-0776 PubMed DOI

Tian CF, Wang ET, Wu LJ, Han TX, Chen WF, Gu CT, Gu JG, Chen WX (2008) Rhizobium fabae sp. nov., a bacterium that nodulates Vicia faba. Int J Syst Evol Microbiol 58:2871–2875. https://doi.org/10.1099/ijs.0.2008/000703-0 PubMed DOI

Zaheer A, Mirza BS, Mclean JE, Yasmin S, Shah TM, Malik KA, Mirza MS (2016) Association of plant growth-promoting Serratia spp. with the root nodules of chickpea. Res Microbiol 167:510–520. https://doi.org/10.1016/j.resmic.2016.04.001 PubMed DOI

Zahir ZA, Zafar-ul-Hye M, Sajjad S, Naveed M (2011) Comparative effectiveness of Pseudomonas and Serratia sp. containing ACC-deaminase for coinoculation with Rhizobium leguminosarum to improve growth, nodulation, and yield of lentil. Biol Fertil Soils 47:457–465. https://doi.org/10.1007/s00374-011-0551-7 DOI

Zhang XX, Kosier B, Priefer UB (2001) Genetic diversity of indigenous Rhizobium leguminosarum bv. viciae isolates nodulating two different host plants during soil restoration with alfalfa. Mol Ecol 10:2297–2305. https://doi.org/10.1046/j.0962-1083.2001.01364.x PubMed DOI

Zhang YJ, Zheng WT, Everall I, Young JPW, Zhang XX, Tian CF, Sui XH, Wang ET, Chen WX (2015) Rhizobium anhuiense sp. nov., isolated from effective nodules of Vicia faba and Pisum sativum. Int J Syst Evol Microbiol 65:2960–2967. https://doi.org/10.1099/ijs.0.000365 PubMed DOI

Zhang J, Shang Y, Peng S, Chen W, Wang E, de Lajudie P, Li B, Guo C, Liu C (2019) Rhizobium sophorae, Rhizobium laguerreae, and two novel Rhizobium genospecies associated with Vicia sativa L. in Northwest China. Plant Soil 442:113–126. https://doi.org/10.1007/s11104-019-04168-w DOI

Zhang J, Li S, Wang N, Yang T, Brunel B, Andrews M, Zong X, Wang E (2022) Rhizobium sophorae is the dominant rhizobial symbiont of Vicia faba L. In North China Syst Appl Microbiol 45:126291. https://doi.org/10.1016/j.syapm.2021.126291 PubMed DOI