Skip to main navigation menu Skip to main content Skip to site footer
Plant Science Today (PST)

Research Articles

Vol. 12 No. 3 (2025)

Morpho-physiochemical and genotypic characterization of rhizobia from chickpea (Cicer arietinum) root nodules

DOI
https://doi.org/10.14719/pst.8876
Submitted
15 April 2025
Published
25-07-2025 — Updated on 06-08-2025
Versions

Abstract

Rhizobia can enhance legume crop productivity, reduce nitrogen fertilizer use and promote sustainable agriculture. This study focused on isolating, identifying and characterizing rhizobia strains from chickpea root nodules. Forty strains were isolated from chickpea root nodules collected from nine districts in Bangladesh, which were cultured on Congo red yeast mannitol agar medium to obtain single colonies and study their morpho-physiological and genetic characteristics. Various physicochemical tests, including gram-staining, bromothymol blue test, temperature, salt and pH tolerance, phosphate solubilization and indole-3-acetic acid (IAA) production tests, were conducted using the
isolated strains. These strains formed milky white colonies with spherical convex surfaces with 1.77 to 8.33 mm diameters. Physicochemical results indicated that strains were gram-negative, fast-growing and acid producers. Maximum strains thrived at pH levels 6 to 8, temperatures ranging from 28 to 35 ºC and 5 % sodium chloride (NaCl). Seven strains exhibited strong phosphate solubilization capabilities and 30 % performed better in IAA production. In aseptic conditions, the inoculated plants showed higher dry matter percentage and higher nitrogen concentration in shoot than non-inoculated ones. Nodulation-positive strains possess nifH gene, indicating nitrogen-fixing capability of these strains. High genetic diversity was observed among the studied rhizobial strains, as determined by enterobacterial repetitive intergenic consensus sequence-based fingerprinting and they formed two major groups. Two strains, CRB-20 and CRB-28, were more diverse than others. Rhizobia isolated from different regions varied in morpho-physiological, genetic characters and symbiotic performances. These strains can be used for field trials to assess their suitability for efficient biological nitrogen fixation and growth of chickpeas.

References

  1. 1. Pawlak K, Kołodziejczak M. The role of agriculture in ensuring food security in developing countries: Considerations in the context of the problem of sustainable food production. Sustainability. 2020;12(13):5488. https://doi.org/10.3390/su12135488
  2. 2. Kumari SG, Vanleur JAG. Viral diseases infecting faba bean (Vicia faba L.). Grain Legumes. 2011;56:24–26.
  3. 3. Gaur PM, Tripathi S, Gowda CLL, Ranga Rao GV, Sharma HC, Pande S, et al. Chickpea seed production manual. Patancheru (India): ICRISAT; 2010. Technical report. http://oar.icrisat.org/id/eprint/10276
  4. 4. Shahbandeh M. Volume of chickpeas produced worldwide in 2022, by country [Internet]. Statista; 2024. Available from: https://www.statista.com/statistics/722203/chickpeas-production-volume-by-country-worldwide/
  5. 5. Peix A, Rivas-Boyero AA, Mateos PF, Rodriguez-Barrueco C, Martõanez-Molina E, Velazquez E. Growth promotion of chickpea and barley by a phosphate solubilizing strain of Mesorhizobium mediterraneum under growth chamber conditions. Soil Biol Biochem. 2001;33(1):103–10. https://doi.org/10.1016/S0038-0717(00)00120-6
  6. 6. Wadhwa Z, Srivastava V, Rani R, Tanvi, Makkar K, Jangra S. Isolation and characterization of rhizobia from chickpea (Cicer arietinum). Int J Curr Microbiol Appl Sci. 2017;6(11):2880–93. https://doi.org/10.20546/ijcmas.2017.611.340
  7. 7. Yesmin MM, Rashid MHO, Prosun TA, Ali MY, Haider MJ, Paul SK. Isolation and characterization of rhizobia strains from root nodules of faba bean (Vicia faba L.). J Bangladesh Agric Univ. 2021;19(3):302–09. https://doi.org/10.5455/JBAU.86499
  8. 8. Savci S. Investigation of effect of chemical fertilizers on environment. APCBEE Procedia. 2012;1:287–92. https://doi.org/10.1016/j.apcbee.2012.03.047
  9. 9. Simon Z, Kelvin M, Amare G, Patrick AN. Isolation and characterization of nitrogen-fixing rhizobia from cultivated and uncultivated soils of Northern Tanzania. Am J Plant Sci. 2014;5(26):4050–67. https://doi.org/10.4236/ajps.2014.526423
  10. 10. Hossain Z, Wang X, Hamel C, Knight JD, Morrison MJ, Gan Y. Biological nitrogen fixation by pulse crops on semiarid Canadian prairies. Can J Plant Sci. 2016;97(1):119–31. https://doi.org/10.1139/cjps-2016-0185
  11. 11. Gebremariam M, Tesfay T. Effect of P application rate and rhizobia inoculation on nodulation, growth, and yield performance of chickpea (Cicer arietinum L.). Int J Agron. 2021;2021(5):1–14. https://doi.org/10.1155/2021/8845489
  12. 12. Walley FL, Kyei-Boahen S, Hnatowich G, Stevenson C. Nitrogen and phosphorus fertility management for desi and kabuli chickpea. Can J Plant Sci. 2005;85(1):73–79. https://doi.org/10.4141/P04-039
  13. 13. Berrada H, Bendrahim KF. Taxonomy of the rhizobia: Current perspectives. Br Microbiol Res J. 2014;4(6):616–39. https://doi.org/10.9734/BMRJ/2014/5635
  14. 14. Laranjo M, Branco C, Soares R, Alho L, Carvalho MD, Oliveira S. Comparison of chickpea rhizobia isolates from diverse Portuguese natural populations based on symbiotic effectiveness and DNA fingerprint. J Appl Microbiol. 2002;92(6):1043–50. https://doi.org/10.1046/j.1365-2672.2002.01615.x
  15. 15. Agrawal PK, Agrawal S, Singh U, Katiyar N, Verma SK. Phenotypic characterization of rhizobia from legumes and its application as a bioinoculant. J Agric Technol. 2012;8(2):681–92. https://api.semanticscholar.org/CorpusID:34346868
  16. 16. Ligiane AF, Pedro M, Jacqueline SS, Karina BS, Fatima M. Diversity and efficiency of Bradyrhizobium strains isolated from soil samples collected from around Sesbania virgata roots using cowpea as trap species. Rev Bras Cienc Solo. 2010;34(4):1113–23. https://doi.org/10.1590/S0100-06832010000400011
  17. 17. Laranjo M, Alexandre A, Oliveira S. Legume growth-promoting rhizobia: An overview on the Mesorhizobium genus. Microbiol Res. 2014;169(1):2–17. https://doi.org/10.1016/j.micres.2013.09.012
  18. 18. Ogutcu H, Adiguzel A, Gulluce M, Karadayi M, Sahin F. Molecular characterization of Rhizobium strains isolated from wild chickpeas collected from high altitudes in Erzurum, Turkey. Rom Biotechnol Lett. 2009;14(2):4294–300. https://hdl.handle.net/20.500.12513/2216
  19. 19. Giongo A, Ambrosini A, Vargas LK, Freire JRJ, Bodanese-Zanettini MH, Passaglia LMP. Evaluation of genetic diversity of Bradyrhizobia strains nodulating soybean (Glycine max L. Merrill) isolated from South Brazilian fields. Appl Soil Ecol. 2008;38:261–69. https://doi.org/10.1016/j.apsoil.2007.10.016
  20. 20. Marzec-Grządziel A, Gałązka A, Marek-Kozaczuk M, Skorupska A. Genetic and phenotypic diversity of rhizobia isolated from Trifolium rubens root nodules. Agronomy. 2020;10(9):1286. https://doi.org/10.3390/agronomy10091286
  21. 21. Somasegaran P, Hoben HJ. Handbook for rhizobia: Methods in legume rhizobia technology. Heidelberg (DE): Springer; 1994. https://doi.org/10.1007/978-1-4613-8375-8
  22. 22. Roychowdhury D, Paul M, Banerjee SK. Isolation, identification and characterization of bacteria (rhizobia) from chickpea (Cicer arietinum) and production of biofertilizer. Eur J Biotechnol Biosci. 2015;3(12):26–29. https://www.researchgate.net/publication/301799385
  23. 23. Nautiyal CS. An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol Lett. 1999;170(1):265–70. https://doi.org/10.1111/j.1574-6968.1999.tb13383.x
  24. 24. Mehta S, Nautiyal CS. An efficient method for qualitative screening of phosphate-solubilizing bacteria. Curr Microbiol. 2001;43(1):51–56. https://doi.org/10.1007/s002840010259
  25. 25. Rahman A, Sitepu IR, Tang SY, Hashidoko Y. Salkowski’s reagent test as a primary screening index for functionalities of rhizobacteria isolated from wild dipterocarp saplings growing naturally on medium-strongly acidic tropical peat soil. Biosci Biotechnol Biochem. 2010;74(11):2202–208. https://doi.org/10.1271/bbb.100360
  26. 26. Fåhreus G. The infection of clover root hairs by nodule bacteria was studied by a simple glass slide technique. Microbiology. 1957;16(2):374–81. https://doi.org/10.1099/00221287-16-2-374
  27. 27. Rashid MH. Genetic diversity of rhizobia nodulating lentils (Lens culinaris Medik) [PhD Thesis]. Heidelberg (DE): Ruperto-Carola University of Heidelberg; 2013.
  28. 28. Nelson DW, Sommers LE. Determination of total nitrogen in plant material. Agron J. 1973;65(1):109–12. https://doi.org/10.2134/agronj1973.00021962006500010033x
  29. 29. Chen WP, Kuo TT. A simple and rapid method for the preparation of gram-negative bacterial genomic DNA. Nucleic Acids Res. 1993;21(9):2260. https://doi.org/10.1093/nar/21.9.2260
  30. 30. Poly F, Monrozier LJ, Bally R. Improvement in the RFLP procedure for studying the diversity of nifH genes in communities of nitrogen fixers in soil. Res Microbiol. 2001;152(1):95–103. https://doi.org/10.1016/S0923-2508(00)01172-4
  31. 31. De Bruijn FJ. 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. 1992;58(7):2180–87. https://doi.org/10.1128/aem.58.7.2180-2187.1992
  32. 32. Wang JY, Wang R, Zhang YM, Liu HC, Chen WF, Wang ET, et al. Bradyrhizobium daqingense sp. nov., isolated from soybean nodules. Int J Syst Evol Microbiol. 2013;63(2):616–24. https://doi.org/10.1099/ijs.0.034280-0
  33. 33. Wubie G, Adal M. Isolation and characterization of chickpea (Cicer arietinum L.) nodulating rhizobia collected from South Wollo Zone, Ethiopia. Int J Agron. 2021;2021(2):1–11. https://doi.org/10.1155/2021/7938399
  34. 34. Damtew ZM. Morphological variation and basic characteristics of selected indigenous rhizobia isolated from major chickpea (Cicer arietinum L.) growing regions of Ethiopia. J Plant Pathol Microbiol. 2021;12(11):585.
  35. 35. Garg N, Singla R. Variability in the response of chickpea cultivars to short-term salinity, in terms of water retention capacity, membrane permeability and osmo-protection. Turk J Agric For. 2009;33(1):57–63. https://doi.org/10.3906/tar-0712-41
  36. 36. Vanderlinde EM, Harrison JJ, Muszynski A, Carlson RW, Turner RJ, Yost CK. Identification of a novel ABC transporter required for desiccation tolerance and biofilm formation in Rhizobium leguminosarum cv. viciae. FEMS Microbiol Ecol. 2010;71(3):327–40. https://doi.org/10.1111/j.1574-6941.2009.00824.x
  37. 37. Aynalem B, Temesgen T, Bezie Y, Yohannis SW. Phenotypic characterization and symbiotic effectiveness test of chickpea (Cicer arietinum L.) rhizobia isolated from Dejen and Aneded districts, East Gojjam zone, Amhara region, Ethiopia. Afr J Biotechnol. 2018;17(23):730–38. https://doi.org/10.5897/AJB2018.16434
  38. 38. Oparah IA, Hartley JC, Deaker R, Gemell G, Hartley E, Kaiser BN. Symbiotic effectiveness, abiotic stress tolerance and phosphate solubilizing ability of new chickpea root-nodule bacteria from soils in Kununurra Western Australia and Narrabri New South Wales Australia. Plant Soil. 2024;495(1):371-89. https://doi.org/10.1007/s11104-023-06331-w
  39. 39. Kenasa G, Jida M, Assefa F. Characterization of phosphate solubilizing faba bean (Vicia faba L.) nodulating rhizobia isolated from acidic soils of Wollega, Ethiopia. Sci Technol Arts Res J. 2014;3(3):11–17. http://dx.doi.org/10.4314/star.v3i3.2
  40. 40. Mir MI, Kumar BK, Gopalakrishnan S, Vadlamudi S, Hameeda B. Characterization of rhizobia isolated from leguminous plants and their impact on the growth of ICCV 2 variety of chickpea (Cicer arietinum L.). Heliyon. 2021;7(11):e08321. https://doi.org/10.1016/j.heliyon.2021.e08321
  41. 41. Sindhu S, Jangra M, Dahiya A, Gera R. Exploration of chickpea rhizobia for solubilization potential of phosphorus, zinc, and potassium. Pharma Innov J. 2019;8(9):426–29.
  42. 42. Kumar SC, Kumar M, Singh R, Saxena AK. Population and genetic diversity of rhizobia nodulating chickpea in Indo-Gangetic plains of India. Braz J Microbiol. 2024;55(4):4057–75. https://doi.org/10.1007/s42770-024-01473-0
  43. 43. Widawati S. Isolation of Indole Acetic Acid (IAA) producing Bacillus siamensis from peat and optimization of the culture conditions for maximum IAA production. In: IOP Conference Series: Earth and Environmental Science. Bristol: IOP Publishing; 2020;572(1):012025. https://doi.org/10.1088/1755-1315/572/1/012025
  44. 44. Zafar M, Ahmed N, Mustafa G, Zahir ZA, Simms EL. Molecular and biochemical characterization of rhizobia from chickpea (Cicer arietinum). Pak J Agric Sci. 2017;54(2):373–81. https://doi.org/10.21162/pakjas/17.5874
  45. 45. Lata DL, Abdie O, Rezene Y. IAA-producing bacteria from the rhizosphere of chickpea (Cicer arietinum L.): Isolation, characterization, and their effects on plant growth performance. Heliyon. 2024;10(21):e39702. https://doi.org/10.1016/j.heliyon.2024.e39702
  46. 46. Ansari M, Malakouti MJ, Rejali F, Mokhtassi Bidgoli A, Golkari S. Screening and identification of the most effective rhizobial isolate (Mezorhizobium ciceri) and investigating its interaction with arbuscular mycorrhizal fungus in the yield and quality of chickpea seeds of Anna. J Soil Bio. 2025;12(2):261-77.
  47. 47. Sindhu SS, Sharma R, Sindhu S, Sehrawat A. Soil fertility improvement by symbiotic rhizobia for sustainable agriculture. In: Panpatte D, Jhala Y, editors. Soil fertility management for sustainable development. Singapore: Springer; 2019. p. 201–24. https://doi.org/10.1007/978-981-13-5904-0_7
  48. 48. Bogino P, Banchio E, Carlos B, Walter G. Competitiveness of a Bradyrhizobium sp. strain in soils containing indigenous rhizobia. Curr Microbiol. 2008;56(1):66–72. https://doi.org/10.1007/s00284-007-9041-4
  49. 49. Nandwani R, Dudeja SS. Molecular diversity of a native mesorhizobial population nodulating chickpea (Cicer arietinum L.) in Indian soils. J Basic Microbiol. 2009;49(5):463–70. https://doi.org/10.1002/jobm.200800355
  50. 50. Belechheb T, Yemalahi A, Bouhnik O, Hassani MZ, El Galiou O, Laglaoui A, et al. Plant growth of the wild forage legume Genista monspessulana is improved by Bradyrhizobium sp. sv. genistearum in the acidic soils of Northern Morocco. Curr Microbiol. 2025;82(6):267. https://doi.org/10.1007/s00284-025-04249-3

Downloads

Download data is not yet available.