Skip to main content
SpringerLink
Log in

Pangenomes-identified singletons for designing specific primers to identify bacterial strains in a plant growth-promoting consortium

  • Original Article
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Background

The use of plant growth-promoting microorganisms represents a sustainable way to increase agricultural yields and plant health. Thus, the identification and tracking of these microorganisms are determinants for validating their positive effects on crops. Pangenomes allow the identification of singletons that can be used to design specific primers for the detection of the studied strains.

Objective

This study aimed to establish a strategy based on the use of whole-genome sequencing and pangenomes for designing and validating primer sets for detecting Bacillus cabrialesii TE3T, Priestia megaterium TRQ8, and Bacillus paralicheniformis TRQ65, a promising beneficial bacterial consortium for wheat.

Methods and Results

The identification of singletons of TE3T, TRQ8, and TRQ65 was performed by pangenomes using the Kbase platform and subsequently analyzed using BLAST®. The identified DNA regions were used for primer design in AlleleID version 7. Primers were validated by multiplex PCR using pure template DNA from each studied strain, combinations of two or three DNA from these strains, and DNA from agricultural soil samples enriched (and not) with the bacterial consortium. Here, we report the first design of primers capable of detecting and identifying the beneficial strains TE3T, TRQ8, and TRQ65.

Conclusions

The use of pangenomes allowed the distinction of unique sequences that enables the design of primers for specific identification of the studied bacterial strains. This strategy can be widely used for the design of primer sets to detect other strains of interest for combating biopiracy, and commercial protection of biological products, among other applications.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Lopez E, Hertel T (2021) Global food waste across the income spectrum: Implications for food prices, production and resource use. Food Policy 98:101874. https://doi.org/10.1016/j.foodpol.2020.101874

    Article  Google Scholar 

  2. de los Santos VS, Parra Cota FI, Herrera Sepúlveda A, Valenzuela Aragón B, Estrada Mora JC (2018) Colmena: colección de microorganismos edáficos y endófitos nativos para contribuir a la seguridad alimentaria nacional. Revista Mexicana de Ciencias Agrícolas. 9:191–202. https://doi.org/10.29312/remexca.v9i1.858

    Article  Google Scholar 

  3. de los Santos-Villalobos S, Díaz-Rodríguez AM, Ávila-Mascareño MF, Martínez-Vidales AD, Parra-Cota FI (2021) COLMENA: a culture collection of native microorganisms for harnessing the agro-biotechnological potential in soils and contributing to food security. Diversity (Basel) 13:337. https://doi.org/10.3390/d13080337

    Article  CAS  Google Scholar 

  4. de los Santos-Villalobos S, Robles RI, Parra FI, Larsen J, Lozano P, Tiedje J (2019) Bacillus cabrialesii sp. nov., an endophytic plant growth promoting bacterium isolated from wheat (Triticum turgidum subsp. durum) in the Yaqui Valley, Mexico. Int J System Evolut Microbiol 69:3939–3945. https://doi.org/10.1099/ijsem.0.003711

    Article  CAS  Google Scholar 

  5. Robles-Montoya RI, Parra-Cota FI, de los Santos-Villalobos S (2019) Draft genome sequence of Bacillus megaterium TRQ8, a plant growth-promoting bacterium isolated from wheat (Triticum turgidum subsp. durum) rhizosphere in the Yaqui Valley, Mexico. 3 Biotech. https://doi.org/10.1007/s13205-019-1726-4

    Article  PubMed  PubMed Central  Google Scholar 

  6. Valenzuela-Ruiz V, Robles-Montoya RI, Parra-Cota FI, Santoyo G, del Carmen Orozco-Mosqueda M, Rodríguez-Ramírez R, de los Santos-Villalobos S (2019) Draft genome sequence of Bacillus paralicheniformis TRQ65, a biological control agent and plant growth-promoting bacterium isolated from wheat (Triticum turgidum subsp. durum) rhizosphere in the Yaqui Valley, Mexico. 3 Biotech. https://doi.org/10.1007/s13205-019-1972-5

    Article  PubMed  PubMed Central  Google Scholar 

  7. Robles-Montoya RI, Chaparro-Encinas LA, Parra-Cota FI, de los Santos VS (2020) Mejorando rasgos biométricos de plántulas de trigo con la inoculación de un consorcio nativo de Bacillus. Revista Mexicana de Ciencias Agrícolas 11:229–235. https://doi.org/10.29312/remexca.v11i1.2162

    Article  Google Scholar 

  8. Rojas-Padilla J, Chaparro-Encinas LA, Robles-Montoya RI, de los Santos VS (2020) Promoción de crecimiento en trigo (Triticum turgidum L. subsp. durum) por la co-inoculación de cepas nativas de Bacillus aisladas del Valle del Yaqui México. Nova Scientia. https://doi.org/10.21640/ns.v12i24.2136

    Article  Google Scholar 

  9. Villa-Rodríguez E, Parra-Cota F, Castro-Longoria E, López-Cervantes J, de los Santos-Villalobos S (2019) Bacillus subtilis TE3: a promising biological control agent against Bipolaris sorokiniana, the causal agent of spot blotch in wheat (Triticum turgidum L. subsp. durum). Biol Control 132:135–143. https://doi.org/10.1016/j.biocontrol.2019.02.012

    Article  CAS  Google Scholar 

  10. Villa-Rodriguez E, Moreno-Ulloa A, Castro-Longoria E, Parra-Cota FI, de los Santos-Villalobos S (2021) Integrated omics approaches for deciphering antifungal metabolites produced by a novel Bacillus species, B. cabrialesii TE3T, against the spot blotch disease of wheat (Triticum turgidum L. subsp. durum). Microbiol Res 251:126826. https://doi.org/10.1016/j.micres.2021.126826

    Article  CAS  PubMed  Google Scholar 

  11. Ibarra-Villarreal AL, Gándara-Ledezma A, Godoy-Flores AD, Herrera-Sepúlveda A, Díaz-Rodríguez AM, Parra-Cota FI, de los Santos-Villalobos S (2021) Salt-tolerant Bacillus species as a promising strategy to mitigate the salinity stress in wheat (Triticum turgidum subsp durum). J Arid Environ 186:104399. https://doi.org/10.1016/j.jaridenv.2020.104399

    Article  Google Scholar 

  12. Baudoin E, Couillerot O, Spaepen S, Moënne-Loccoz Y, Nazaret S (2010) Applicability of the 16S–23S rDNA internal spacer for PCR detection of the phytostimulatory PGPR inoculant Azospirillum lipoferum CRT1 in field soil. J Appl Microbiol 108:25–38. https://doi.org/10.1111/j.1365-2672.2009.04393.x

    Article  CAS  PubMed  Google Scholar 

  13. Atkins SD, Clark IM, Pande S, Hirsch PR, Kerry BR (2005) The use of real-time PCR and species-specific primers for the identification and monitoring of Paecilomyces lilacinus. FEMS Microbiol Ecol 51:257–264. https://doi.org/10.1016/j.femsec.2004.09.002

    Article  CAS  PubMed  Google Scholar 

  14. Couillerot O, Bouffaud M-L, Baudoin E, Muller D, Caballero-Mellado J, Moënne-Loccoz Y (2010) Development of a real-time PCR method to quantify the PGPR strain Azospirillum lipoferum CRT1 on maize seedlings. Soil Biol Biochem 42:2298–2305. https://doi.org/10.1016/j.soilbio.2010.09.003

    Article  CAS  Google Scholar 

  15. Mendis HC, Thomas VP, Schwientek P, Salamzade R, Chien J-T, Waidyarathne P, Kloepper J, de La Fuente L (2018) Strain-specific quantification of root colonization by plant growth promoting rhizobacteria Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 in non-sterile soil and field conditions. PLoS ONE 13:e0193119. https://doi.org/10.1371/journal.pone.0193119

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ruppel S, Rühlmann J, Merbach W (2006) Quantification and localization of bacteria in plant tissues using quantitative real-time PCR and online emission fingerprinting. Plant Soil 286:21–35. https://doi.org/10.1007/s11104-006-9023-5

    Article  CAS  Google Scholar 

  17. Stets MI, Campbell Alqueres SM, Maltempi Souza E, de Oliveira PF, Schmid M, Hartmann A, Magalhães Cruz L (2015) Quantification of Azospirillum brasilense FP2 bacteria in wheat roots by strain-specific quantitative PCR. Appl Environ Microbiol 81:6700–6709. https://doi.org/10.1128/AEM.01351-15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zhang S, Ma Y, Jiang W, Meng L, Cao X, Hu J, Chen J, Li J (2020) Development of a strain-specific quantification method for monitoring Bacillus amyloliquefaciens TF28 in the rhizospheric soil of soybean. Mol Biotechnol 62:521–533. https://doi.org/10.1007/s12033-020-00268-6

    Article  CAS  PubMed  Google Scholar 

  19. Zhang Y, Gao X, Wang S, Zhu C, Li R, Shen Q (2018) Application of Bacillus velezensis NJAU-Z9 enhanced plant growth associated with efficient rhizospheric colonization monitored by qPCR with primers designed from the whole genome sequence. Curr Microbiol 75:1574–1583. https://doi.org/10.1007/s00284-018-1563-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Urrea-Valencia S, Etto RM, Takahashi WY, Caires EF, Bini AR, Ayub RA, Stets MI, Cruz LM, Galvão CW (2021) Detection of Azospirillum brasilense by qPCR throughout a maize field trial. Appl Soil Ecol 160:103849. https://doi.org/10.1016/j.apsoil.2020.103849

    Article  Google Scholar 

  21. Guimarães LC, Benevides de Jesus L, Canario Viana MV, Artur S, Juca Ramos RT, de Castro SS, Azevedo V (2015) Inside the pan-genome - methods and software overview. Curr Genom 16:245–252. https://doi.org/10.2174/1389202916666150423002311

    Article  CAS  Google Scholar 

  22. Arkin AP, Cottingham RW, Henry CS, Harris NL, Stevens RL, Maslov S, Dehal P, Ware D, Perez F, Canon S, Sneddon MW, Henderson ML, Riehl WJ, Murphy-Olson D, Chan SY, Kamimura RT, Kumari S, Drake MM, Brettin TS, Glass EM, Chivian D, Gunter D, Weston DJ, Allen BH, Baumohl J, Best AA, Bowen B, Brenner SE, Bun CC, Chandonia J-M, Chia J-M, Colasanti R, Conrad N, Davis JJ, Davison BH, DeJongh M, Devoid S, Dietrich E, Dubchak I, Edirisinghe JN, Fang G, Faria JP, Frybarger PM, Gerlach W, Gerstein M, Greiner A, Gurtowski J, Haun HL, He F, Jain R, Joachimiak MP, Keegan KP, Kondo S, Kumar V, Land ML, Meyer F, Mills M, Novichkov PS, Oh T, Olsen GJ, Olson R, Parrello B, Pasternak S, Pearson E, Poon SS, Price GA, Ramakrishnan S, Ranjan P, Ronald PC, Schatz MC, Seaver SMD, Shukla M, Sutormin RA, Syed MH, Thomason J, Tintle NL, Wang D, Xia F, Yoo H, Yoo S, Yu D (2018) KBase: the united states department of energy systems biology knowledgebase. Nat Biotechnol 36:566–569. https://doi.org/10.1038/nbt.4163

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Andrews S (2010) FastQC: a quality control tool for high throughput sequence data. In: http://www.bioinformatics.babraham.ac.uk/projects/fastqc

  24. Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. https://doi.org/10.1093/bioinformatics/btu170

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. https://doi.org/10.1089/cmb.2012.0021

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Darling ACE, Mau B, Blattner FR, Perna NT (2004) Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res 14:1394–1403. https://doi.org/10.1101/gr.2289704

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Yoon S-H, Ha S-M, Kwon S, Lim J, Kim Y, Seo H, Chun J (2017) Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int J Syst Evol Microbiol 67:1613–1617. https://doi.org/10.1099/ijsem.0.001755

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Blom J, Albaum SP, Doppmeier D, Pühler A, Vorhölter F-J, Zakrzewski M, Goesmann A (2009) EDGAR: a software framework for the comparative analysis of prokaryotic genomes. BMC Bioinform. https://doi.org/10.1186/1471-2105-10-154

    Article  Google Scholar 

  29. Tettelin H, Medini D (2020) The pangenome diversity, dynamics and evolution of genomes. Springer Nature Switzerland AG, Cham

    Book  Google Scholar 

  30. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410. https://doi.org/10.1016/S0022-2836(05)80360-2

    Article  CAS  PubMed  Google Scholar 

  31. Valenzuela-Aragon B, Parra-Cota FI, Santoyo G, Arellano-Wattenbarger GL, de los Santos-Villalobos S (2019) Plant-assisted selection: a promising alternative for in vivo identification of wheat (Triticum turgidum L. subsp. durum) growth promoting bacteria. Plant Soil 435:367–384. https://doi.org/10.1007/s11104-018-03901-1

    Article  CAS  Google Scholar 

  32. Raeder U, Broda P (1985) Rapid preparation of DNA from filamentous fungi. Lett Appl Microbiol 1:17–20. https://doi.org/10.1111/j.1472-765X.1985.tb01479.x

    Article  CAS  Google Scholar 

  33. Bresler MM, Rosser SJ, Basran A, Bruce NC (2000) Gene cloning and nucleotide sequencing and properties of a cocaine esterase from Rhodococcus sp. Strain MB1. Appl Environ Microbiol 66:904–908. https://doi.org/10.1128/AEM.66.3.904-908.2000

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Bustin S, Huggett J (2017) qPCR primer design revisited. Biomol Detect Quantif 14:19–28. https://doi.org/10.1016/j.bdq.2017.11.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Navarro E, Serrano-Heras G, Castaño MJ, Solera J (2015) Real-time PCR detection chemistry. Clin Chim Acta 439:231–250. https://doi.org/10.1016/j.cca.2014.10.017

    Article  CAS  PubMed  Google Scholar 

  36. Rilling JI, Acuña JJ, Nannipieri P, Cassan F, Maruyama F, Jorquera MA (2019) Current opinion and perspectives on the methods for tracking and monitoring plant growth-promoting bacteria. Soil Biol Biochem 130:205–219. https://doi.org/10.1016/j.soilbio.2018.12.012

    Article  CAS  Google Scholar 

  37. Sivashankari S, Shanmughavel P (2007) Comparative genomics - a perspective. Bioinformation 1:376–378. https://doi.org/10.6026/97320630001376

    Article  PubMed  PubMed Central  Google Scholar 

  38. Tettelin H, Masignani V, Cieslewicz MJ, Donati C, Medini D, Ward NL, Angiuoli SV, Crabtree J, Jones AL, Durkin AS, DeBoy RT, Davidsen TM, Mora M, Scarselli M, Margarit y Ros I, Peterson JD, Hauser CR, Sundaram JP, Nelson WC, Madupu R, Brinkac LM, Dodson RJ, Rosovitz MJ, Sullivan SA, Daugherty SC, Haft DH, Selengut J, Gwinn ML, Zhou L, Zafar N, Khouri H, Radune D, Dimitrov G, Watkins K, O’Connor KJB, Smith S, Utterback TR, White O, Rubens CE, Grandi G, Madoff LC, Kasper DL, Telford JL, Wessels MR, Rappuoli R, Fraser CM (2005) Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: Implications for the microbial “pan-genome.” Proc Natl Acad Sci 102:13950–13955. https://doi.org/10.1073/pnas.0506758102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Reddypriya P, Soumare A, Balachandar D (2019) Multiplex and quantitative PCR targeting SCAR markers for strain-level detection and quantification of biofertilizers. J Basic Microbiol 59:111–119. https://doi.org/10.1002/jobm.201800318

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Amelia C. Montoya-Martínez acknowledges Consejo Nacional de Ciencia y Tecnología (CONACYT) for funding postdoctoral stay (application number 866971).

Funding

This study was supported by Instituto Tecnológico de Sonora (ITSON) through the PROFAPI project 2022-0001.

Author information

Author notes

  1. Roel Alejandro Chávez-Luzanía and Amelia C. Montoya-Martínez contributed equally to the research.

Authors and Affiliations

  1. Instituto Tecnológico de Sonora. 5 de febrero 818 sur, CP. 85000, Cd. Obregón, SON, México

    Roel Alejandro Chávez-Luzanía, Amelia C. Montoya-Martínez & Sergio de los Santos-Villalobos

  2. Campo Experimental Norman E. Borlaug, Instituto Nacional de Investigaciones forestales, Agrícolas y Pecuarias, Norman E. Borlaug Km. 12, C.P. 85000, Ciudad Obregón, SON, México

    Fannie Isela Parra-Cota

Authors
  1. Roel Alejandro Chávez-Luzanía
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Amelia C. Montoya-Martínez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fannie Isela Parra-Cota
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sergio de los Santos-Villalobos
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

SdlS-V and FIP-C: The conceptualization of the study, methodology design, validation, formal analysis, and revisions of the manuscript were carried out. RAC-L and AC-M: The methodology design, validation, formal analysis, data curation, and writing of the original draft were carried out.

Corresponding author

Correspondence to Sergio de los Santos-Villalobos.

Ethics declarations

Conflict of interest

The authors have declared that there is no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 2404 KB)

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chávez-Luzanía, R.A., Montoya-Martínez, A.C., Parra-Cota, F.I. et al. Pangenomes-identified singletons for designing specific primers to identify bacterial strains in a plant growth-promoting consortium. Mol Biol Rep 49, 10489–10498 (2022). https://doi.org/10.1007/s11033-022-07927-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-022-07927-8

Keywords

Search

Navigation