Isolation and evaluation of endophytic bacteria from root nodules of Glycine max L. (Merr.) and their potential use as biofertilizers

  • Arely A. Vargas-Díaz CONACYT−Colegio de Postgraduados, Champotón, Campus Campeche. Ctra. Federal Haltunchén-Edzna km 17.5. Sihochac, Champotón, 24450 Campeche
  • Ronald Ferrera-Cerrato Colegio de Postgraduados, Posgrado de Edafología. Campus Montecillo, 56230 Texcoco
  • Hilda V. Silva-Rojas Colegio de Postgraduados, Posgrado de Recursos Genéticos y Productividad. Campus Montecillo 56230 Texcoco
  • Alejandro Alarcón Colegio de Postgraduados, Posgrado de Edafología. Campus Montecillo, 56230 Texcoco
Keywords: Bradyrhizobium, co-inoculation, free-living endophytic bacteria, endophytic symbiotic bacteria, plant growth promotion, soybean, symbiosis

Abstract

Aim of study: To isolate and characterize endophytic bacteria inhabiting soybean root nodules collected from two tropical cropping systems in Mexico, and to evaluate the bacterial effects in soybean plants under controlled conditions.

Area of study: The study was carried out at two locations (San Antonio Cayal and Nuevo Progreso municipalities) of Campeche State, Mexico.

Material and methods: Two experimental stages were performed: 1) isolation, morphological and biochemical characterization, and molecular identification of endophytic bacteria from root-nodules of four soybean varieties grown at field conditions; and 2) evaluation of the effects of endophytic isolates on soybean growth and nodule development, and the effects of bacterial co-inoculation on soybean plants, under controlled conditions.

Main results: Twenty-three endophytic bacteria were isolated from root nodules, and identified as Agrobacterium, Bradyrhizobium, Rhizobium, Ensifer, Massilia, Chryseobacterium, Enterobacter, Microbacterium, Serratia, and Xanthomonas. Under controlled conditions, Rhizobium sp. CPO4.13C or Agrobacterium tumefaciens CPO4.15C significantly increased the plant height (46% and 41%, respectively), whereas Bradyrhizobium sp. CPO4.24C promoted the nodule formation (36 nodules/plant). The co-inoculation of B. japonicum USDA110 and Bradyrhizobium sp. CPO4.24C enhanced plant growth, height (33.87 cm), root nodulation (69 nodules/plant) and N-fixation (3.10 µmol C2H4 h-1 plant-1) in comparison to the negative control.

Research highlights:  Results suggest that the native Bradyrhizobium sp. CPO4.24C may be used as a biofertilizer directed to developing sustainable soybean cropping at tropical regions.

Downloads

Download data is not yet available.

References

Abou-Shanab RA, Wongphatcharachai M, Sheaffer CC, Orf JC, Sadowsky MJ, 2017. Competition between introduced Bradyrhizobium japonicum strains and indigenous bradyrhizobia in Minnesota organic farming systems. Symbiosis 73: 155-163. https://doi.org/10.1007/s13199-017-0505-4

Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ, 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389-3404. https://doi.org/10.1093/nar/25.17.3389

Ando S, Yokoyama T, 1999. Phylogenetic analyses of Bradyrhizobium strains nodulating soybean (Glycine max) in Thailand with reference to the USDA strains of Bradyrhizobium. Can J Microbiol 45: 639-645. https://doi.org/10.1139/w99-061

Angulo-Castro A, Ferrera-Cerrato R, Alarcón A, Almaraz-Suárez JJ, Delgadillo-Martínez J, Jiménez-Fernández M, García-Barradas O, 2018. Crecimiento y eficiencia fotoquímica del fotosistema II en plántulas de 2 variedades de Capsicum annuum L. inoculadas con rizobacterias u hongos micorrícicos arbusculares. Rev Argent Microbiol 50 (2): 178-188. https://doi.org/10.1016/j.ram.2017.03.011

Arone G, Calderón C, Moreno S, Bedmar EJ, 2014. Identification of Ensifer strains isolated from root nodules of Medicago hispida grown in association with Zea mays in the Quechua region of the Peruvian Andes. Biol Fertil Soils 50: 185-190. https://doi.org/10.1007/s00374-013-0825-3

Aserse AA, Räsänen LA, Aseffa F, Hailemariam A, Lindström K, 2013. Diversity of sporadic symbionts and nonsymbiotic endophytic bacteria isolated from nodules of woody, shrub, and food legumes in Ethiopia. Appl Microbiol Biotechnol 97: 10117-10134. https://doi.org/10.1007/s00253-013-5248-4

Bai Y, D'Aoust F, Smith DL, Driscoll BT, 2002. Isolation of plant-growth-promoting Bacillus strains from soybean root nodules. Can J Microbiol 48: 230-238. https://doi.org/10.1139/w02-014

Bai Y, Zhou X, Smith DL, 2003. Crop ecology, management and quality: enhanced soybean plant growth resulting from co inoculation of Bacillus strain Bradyrhizobium japonicum. Crop Sci 43: 1774-1781. https://doi.org/10.2135/cropsci2003.1774

Boonkerd N, Singleton P, 2002. Production of Rhizobium biofertilizer. In: Biotechnology of biofertilizers; Kannaiyan S (ed), pp: 122-128. Narosa Publ House, New Delhi, India.

Camacho M, Santamaria C, Temprano F, Rodríguez-Navarro DN, Daza, A, 2001. Co-inoculation with Bacillus sp. CECT 450 improves nodulation in Phaseolus vulgaris L. Can J Microbiol 47: 1058-1062. https://doi.org/10.1139/cjm-47-11-1058

de Carvalho GAB, Batista JSS, Marcelino-Guimarães FC, do Nascimento LC, Hungria, M, 2013. Transcriptional analysis of genes involved in nodulation in soybean roots inoculated with Bradyrhizobium japonicum strain CPAC 15. BMC Genomics 14: 153. https://doi.org/10.1186/1471-2164-14-153

Deng ZS, Zhao LF, Kong ZY, Yang WQ, Lindström K, Wang ET, Wei GH, 2011. Diversity of endophytic bacteria within nodules of the Sphaerophysa salsula in different regions of Loess Plateau in China. FEMS Microbiol Ecol 76: 463-475. https://doi.org/10.1111/j.1574-6941.2011.01063.x

Doyle JJ, Doyle JL, 1990. A rapid total DNA preparation procedure for fresh plant tissue. Focus 12: 13-15. https://doi.org/10.2307/2419362

Eden PA, Schmidt TM, Blakemore RP, Pace NR, 1991. Phylogenetic analysis of Aquaspirillum magnetotacticum using polymerase chain reaction-amplified 16S rRNA-specific DNA. Int J Syst Bacteriol 41: 324-325. https://doi.org/10.1099/00207713-41-2-324

Ezzakkioui F, El Mourabit N, Chahboune R, Castellano-Hinojosa A, Bedmar EJ, Barrijal S, 2015. Phenotypic and genetic characterization of rhizobia isolated from Hedysarum flexuosum in Northwest region of Morocco. J Basic Microbiol 55: 830-837. https://doi.org/10.1002/jobm.201400790

Ferrera-Cerrato R, González-Chávez MCA, Rodríguez-Mendoza MN, 1993. Manual de Agromicrobiología. Editorial Trillas S. A. de C. V. 139 pp.

Gai Z, Zhang J, Li C, 2017. Effects of starter nitrogen fertilizer on soybean root activity, leaf photosynthesis and grain yield. PloS One 12: e0174841. https://doi.org/10.1371/journal.pone.0174841

Ghosh PK, De TK, Maiti TK, 2015. Ascorbic acid production in root, nodule and Enterobacter spp. (Gammaproteobacteria) isolated from root nodule of the legume Abrus precatorius L. Biocatal Agric Biotechnol 4: 127-134. https://doi.org/10.1016/j.bcab.2014.11.006

Hall TA, 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41: 95-98.

Htwe AZ, Yamakawa T, 2016. Low-density co-inoculation with Bradyrhizobium japonicum SAY3-7 and Streptomyces griseoflavus P4 promotes plant growth and nitrogen fixation in soybean cultivars. Am J Plant Sci 7: 1652-1661. https://doi.org/10.4236/ajps.2016.712156

Huelsenbeck JP, Ronquist F, 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754-755. https://doi.org/10.1093/bioinformatics/17.8.754

Hung PQ, Annapurna K, 2004. Isolation and characterization of endophytic bacteria in soybean (Glycine sp.). Omonrice 12: 92-101.

Kueneman EA, Root WR, Dashiell KE, Hohenberg J, 1984. Breeding soybeans for the tropics capable of nodulating effectively with indigenous Rhizobium spp. Plant Soil 82: 387-396. https://doi.org/10.1007/BF02184276

Kumar S, Stecher G, Li M, Knyaz C, Tamura K, 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35: 1547-1549. https://doi.org/10.1093/molbev/msy096

Leite J, Fischer D, Rouws LF, Fernandes-Júnior PI, Hofmann A, Kublik S, Schloter M, Xavier GR, Radl V, 2016. Cowpea nodules harbor non-rhizobial bacterial communities that are shaped by soil type rather than plant genotype. Front Plant Sci 7: 2064. https://doi.org/10.3389/fpls.2016.02064

Li JH, Wang ET, Chen WF, Chen WX, 2008. Genetic diversity and potential for promotion of plant growth detected in nodule endophytic bacteria of soybean grown in Heilongjiang province of China. Soil Biol Biochem 40: 238-246. https://doi.org/10.1016/j.soilbio.2007.08.014

Li L, Sinkko H, Montonen L, Wei G, Lindström K, Räsänen LA, 2012. Biogeography of symbiotic and other endophytic bacteria isolated from medicinal. Glycyrrhiza species in China. FEMS Microbiol Ecol 79: 46-68. https://doi.org/10.1111/j.1574-6941.2011.01198.x

Lira Jr. MA, Nascimento LR, Fracetto GG, 2015. Legume-rhizobia signal exchange: promiscuity and environmental effects. Front Microbiol 6: 945. https://doi.org/10.3389/fmicb.2015.00945

Liu J, Wang ET, Chen WX, 2010. Mixture of endophytic Agrobacterium and Sinorhizobium meliloti strains could induce nonspecific nodulation on some woody legumes. Arch Microbiol 192: 229-234. https://doi.org/10.1007/s00203-010-0543-2

Masuda T, Goldsmith, PD, 2009. World soybean production: area harvested, yield, and long-term projections. Int Food and Agribus Man 12: 143-162.

Palaniappan P, Chauhan PS, Saravanan VS, Anandham R, Sa T, 2010. Isolation and characterization of plant growth promoting endophytic bacterial isolates from root nodule of Lespedeza sp. Biol Fertil Soils 46: 807-816. https://doi.org/10.1007/s00374-010-0485-5

Pongsilp N, Teaumroong N, Nuntagij A, Boonkerd N, Sadowsky MJ 2002. Genetic structure of indigenous non-nodulating and nodulating populations of Bradyrhizobium in soils from Thailand. Symbiosis 33: 39-58.

Ronquist F, Huelsenbeck JP, 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572-1574. https://doi.org/10.1093/bioinformatics/btg180

Saïdi S, Mnasri B, Mhamdi R, 2011. Diversity of nodule-endophytic agrobacteria-like strains associated with different grain legumes in Tunisia. Syst Appl Microbiol 34: 524-530. https://doi.org/10.1016/j.syapm.2011.01.009

Santi C, Bogusz D, Franche C, 2013. Biological nitrogen fixation in non-legume plants. Ann Bot 111: 743-767. https://doi.org/10.1093/aob/mct048

Segovia L, Pinero D, Palacios R, Martínez-Romero E, 1991. Genetic structure of a soil population of nonsymbiotic Rhizobium leguminosarum. Appl Environ Microbiol 57: 426-433.

Soe KM, Yamakawa T, 2013. Evaluation of effective Myanmar Bradyrhizobium strains isolated from Myanmar soybean and effects of co-inoculation with Streptomyces griseoflavus P4 for nitrogen fixation. Soil Sci Plant Nutr 59: 361-370. https://doi.org/10.1080/00380768.2013.794437

Somasegaran P, Hoben HJ, 2012. Handbook for rhizobia: methods in legume-Rhizobium technology. Springer Sci & Business Media. 449 pp.

Stacey G, Vodkin L, Parrott WA, Shoemaker RC, 2004. Draft plan for soybean genomics. Nat Sci Found-sponsored workshop report. Plant Physiol 13: 59-70. https://doi.org/10.1104/pp.103.037903

Stajković O, De Meyer S, Miličić B, Willems A, 2009. Isolation and characterization of endophytic non-rhizobial bacteria from root nodules of alfalfa (Medicago sativa L.). Bot Serb 33: 107-114.

Stajkovic O, Delic D, Josic D, Kuzmanovic D, Rasulic N, Knezevic-Vukcevic J, 2011. Improvement of common bean growth by co-inoculation with Rhizobium and plant growth-promoting bacteria. Rom Biotech Lett 16: 5919-5926.

Sundara R, Sinha M, 1963. Organism phosphate solubilizers in soil. Ind J Agr Sci 33: 272-278.

Suzuki Y, Adhikari D, Itoh K, Suyama K, 2014. Effects of temperatura on competition and relative dominance of Bradyrhizobium japonicum and Bradyrhizobium elkanii in the process of soybean nodulation. Plant Soil 374: 915-924. https://doi.org/10.1007/s11104-013-1924-5

Valencia R, Rubén A, Ligarreto M. 2010. Análisis de la interacción soya-cepa (Bradyrhizobium japonicum) x ambiente, en oxisoles de la Orinoquia colombiana. Agron Colomb 28:356-366.

Vincent JM, 1970. A manual for practical study of root nodule bacteria. IBP Handbook No. 15, Blackwell Sci Publ, Oxford, 164 pp.

Waluyo SH, Lie TA, de Vos WM, 2005. Characterisation of soybean rhizobial strains from Java and Sumatra. Indones J Agric Sci 6: 10-19. https://doi.org/10.21082/ijas.v6n1.2005.10-19

Wang LL, Wang ET, Liu J, Li Y, Chen WX, 2006. Endophytic occupation of root nodules and roots of Melilotus dentatus by Agrobacterium tumefaciens. Microb Ecol 52: 436-443. https://doi.org/10.1007/s00248-006-9116-y

Wu LJ, Wang HQ, Wang ET, Chen WX, Tian CF, 2011. Genetic diversity of nodulating and non-nodulating rhizobia associated with wild soybean (Glycine soja Sieb. & Zucc.) in different ecoregions of China. FEMS Microbiol Ecol 76: 439-450. https://doi.org/10.1111/j.1574-6941.2011.01064.x

Yan J, Han XZ, Ji ZJ, Li Y, Wang ET, Xie ZH, Chen WF, 2014. Abundance and diversity of soybean-nodulating rhizobia in black soil are impacted by land use and crop management. Appl Environ Microbiol 80: 5394-5402. https://doi.org/10.1128/AEM.01135-14

Zhang F, Dashti N, Hynes RK, Smith DL, 1996. Plant growth promoting rhizobacteria and soybean [Glycine max (L.) Merr.] nodulation and nitrogen fixation at suboptimal root zone temperatures. Ann Bot 77: 453-460. https://doi.org/10.1006/anbo.1996.0055

Zhang YM, Li Y, Chen WF, Wang ET, Tian CF, Li QQ, Zhang YZ, Sui XH, Chen WX, 2011. Biodiversity and biogeography of rhizobia associated with soybean plants grown in the North China Plain. Appl Environ Microbiol 77: 6331-6342. https://doi.org/10.1128/AEM.00542-11

Published
2019-11-08
How to Cite
Vargas-Díaz, A. A., Ferrera-Cerrato, R., Silva-Rojas, H. V., & Alarcón, A. (2019). Isolation and evaluation of endophytic bacteria from root nodules of Glycine max L. (Merr.) and their potential use as biofertilizers. Spanish Journal of Agricultural Research, 17(3), e1103. https://doi.org/10.5424/sjar/2019173-14220
Section
Soil science