Skip to main content
Log in

Development of a Strain-Specific Quantification Method for Monitoring Bacillus amyloliquefaciens TF28 in the Rhizospheric Soil of Soybean

  • Original Paper
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Bacillus amyloliquefaciens TF28 can be used to control soybean root disease. To assess its commercial potential as a biocontrol agent, it is necessary to develop a strain-specific quantification method to monitor its colonization dynamics in the rhizospheric soil of soybean under field conditions. Based on genomic comparison with the same species in NCBI databases, a strain-unique gene ukfpg was used as molecular marker to develop strain-specific PCR assay. Among three primer pairs, only primer pairs (F2/R2) could specifically differentiate TF28 from other strains of B. amyloliquefaciens with the detection limit of 10 fg and 100 CFU/g for DNA extracted from pure culture and dry soil, respectively. Then, a colony count coupled with PCR assay was used to monitor the population of TF28 in the rhizospheric soil of soybean in the field. The results indicated that TF28 successfully colonized in the rhizospheric soil of soybean. The colonization population of TF28 changed dynamically within the 120-day growth period with high population at the branching (V6) and flowering stages (R2). This study provides an efficient method to quantitatively monitor the colonization dynamics of TF28 in the rhizospheric soil of soybean in the field and demonstrates the potential of TF28 as a biocontrol agent for commercial development.

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

Access this article

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
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Ciancio, A., Pieterse, C. M., & Mercado-Blanco, J. (2016). Editorial: Harnessing useful rhizosphere microorganisms for pathogen and pest biocontrol. Frontiers in Microbiology, 7, 1620.

    Article  Google Scholar 

  2. Compant, S., Duffy, B., Nowak, J., Clement, C., & Barka, E. A. (2005). Use of plant growth promoting bacteria for biocontrol of plant diseases: Principles, mechanisms of action, and future prospects. Applied and Environmental Microbiology, 71, 4951–4959.

    Article  CAS  Google Scholar 

  3. Weller, D. M. (2007). Pseudomonas biocontrol agents of soilborne pathogens: Looking back over 30 years. Phytopathology, 97, 250–256.

    Article  Google Scholar 

  4. Huang, X. F., Chaparro, J. M., Reardon, K. F., Zhang, R., Shen, Q., & Vivanco, J. M. (2014). Rhizosphere interactions: Root exudates, microbes, and microbial communities. Botany, 92, 267–275.

    Article  Google Scholar 

  5. Gans, J., Wolinsky, M., & Dunbar, J. (2005). Computational improvements reveal great bacterial diversity and high metal toxicity in soil. Science, 309, 1387–1390.

    Article  CAS  Google Scholar 

  6. Van Elsas, J. D., Duarte, G. F., Rosado, A. S., & Smalla, K. (1998). Microbiological and molecular biological methods for monitoring microbial inoculants and their effects in the soil environment. Journal of Microbiological Methods, 32, 133–154.

    Article  Google Scholar 

  7. Badosa, E., Moreno, C., & Montesinos, E. (2004). Lack of detection of transfer of ampicillin resistance genes from Bt176 transgenic corn to culturable bacteria under field conditions. FEMS Microbiology Ecology, 48, 169–178.

    Article  CAS  Google Scholar 

  8. Mahaffee, W. F., Bauske, E. M., van Vuurde, J. W. L., van der Wolf, J. M., van den Brink, M., & Kloepper, J. W. (1997). Comparative analysis of antibiotic resistance, immunofluorescent colony staining, and transgenic marker (Bioluminescence) for monitoring the environmental fate of a rhizobacterium. Applied and Environmental Microbiology, 63, 1617–1622.

    Article  CAS  Google Scholar 

  9. Kredics, L., Chen, L. Q., Kedves, O., Rita, B., Hatvani, L., Allaga, H., et al. (2018). Molecular tools for monitoring Trichoderma in agricultural environments. Frontiers in Microbiology, 9, 1599.

    Article  Google Scholar 

  10. Loncaric, I., Donat, C., Antlinger, B., Oberlerchner, J. T., Heissenberger, B., & Moosbeckhofer, R. (2008). Strain-specific detection of two Aureobasidium pullulans strains, fungal biocontrol agents of fire blight by new, developed multiplex-PCR. Journal of Applied Microbiology, 104, 1433–1441.

    Article  CAS  Google Scholar 

  11. Pujol, M., Badosa, E., Cabrefiga, J., & Montesinos, E. (2005). Development of a strain-specific quantitative method for monitoring Pseudomonas fluorescens EPS62e, a novel biocontrol agent of fire blight. FEMS Microbiology Letters, 249, 343–352.

    Article  CAS  Google Scholar 

  12. Daisuke, K., Naruto, F., Tetsuya, S., Kenichi, T., Seiya, T., & Marion, K. S. (2016). Species-specific detection of Mycosphaerella polygonicuspidati as a biological control agent for Fallopia japonica by PCR assay. Molecular Biotechnology, 58, 626–633.

    Article  CAS  Google Scholar 

  13. Chowdhury, S. P., Dietel, K., Rändler, M., Schmid, M., Junge, H., Borriss, R., et al. (2013). Effects of Bacillus amyloliquefaciens FZB42 on lettuce growth and health under pathogen pressure and its impact on the rhizosphere bacterial community. PLoS ONE, 8, e68818.

    Article  Google Scholar 

  14. Kim, Y. G., Kang, H. K., Kwon, K. D., Seo, C. H., Lee, H. B., & Park, Y. (2015). Antagonistic activities of novel peptides from Bacillus amyloliquefaciens PT14 against Fusarium solani and Fusarium oxysporum. Journal of Agricultural and Food Chemistry, 63, 10380–10387.

    Article  CAS  Google Scholar 

  15. Liu, Y. P., Chen, L., Wu, G. W., Feng, H. C., Zhang, G. S., Shen, Q. R., et al. (2017). Identification of root-secreted compounds involved in the communication between cucumber, the beneficial Bacillus amyloliquefaciens, and the soil-borne pathogen Fusarium oxysporum. Molecular Plant-Microbe Interactions, 30, 53–62.

    Article  CAS  Google Scholar 

  16. Zhang, S. M., Sha, C. Q., Wang, Y. X., Li, J., Zhao, X. Y., & Zhang, X. C. (2008). Isolation and characterization of antifungal endophytic bacteria from soybean. Microbiology, 35, 1593–15999.

    Google Scholar 

  17. Zhang, S. M., Wang, Y. X., Meng, L. Q., Li, J., Zhao, X. Y., & Cao, X. (2012). Isolation and characterization of antifungal lipopeptides produced by endophytic Bacillus amyloliquefaciens TF28. African Journal of Microbiology Research, 6, 1747–1755.

    CAS  Google Scholar 

  18. Zhang, S. M., Jiang, W., Li, J., Meng, L. Q., Cao, X., Hu, J. H., et al. (2016). Whole genome shotgun sequence of Bacillus amyloliquefaciens TF28, a biocontrol entophytic bacterium. Standards in Genomic Sciences, 11, 73.

    Article  Google Scholar 

  19. Reva, O. N., Dixelius, C., Meijer, J., & Priest, F. G. (2004). Taxonomic characterization and plant colonizing abilities of some bacteria related to Bacillus amyloliquefaciens and Bacillus subtilis. FEMS Microbiology Ecology, 48, 249–259.

    Article  CAS  Google Scholar 

  20. Sato, N., Seo, G., & Benno, Y. (2014). Development of strain-specific PCR primers for quantitative detection of Bacillus mesentericus strain TO-A in human Feces. Biological and Pharmaceutical Bulletin, 37, 123–129.

    Article  CAS  Google Scholar 

  21. Felici, C., Vettori, L., Toffanin, A., & Nuti, M. (2008). Developmentofa strain-specific genomicmarker formonitoring a Bacillus subtilis biocontrol strain in the rhizosphere of tomato. FEMS Microbiology Ecology, 65, 289–298.

    Article  CAS  Google Scholar 

  22. Johansson, A. H., Bejai, S., Niazi, A., Manzoor, S., Bongcam-Rudloff, E., & Meijer, J. (2014). Studies of plant colonisation by closely related Bacillus amyloliquefaciens biocontrol agents using strain specific quantitative PCR assays. Antonie van Leeuwenhoek, 106, 1247–1257.

    Article  CAS  Google Scholar 

  23. Mendis, H. C., Thomas, V. P., Schwientek, P., Salamzade, R., Chien, J. T., Waidyarathne, P., et al. (2018). Strain-specific quantification of root colonization by plant growth promoting rhizobacteria Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 in nonsterile soil and field conditions. PLoS ONE, 13, e0193119.

    Article  CAS  Google Scholar 

  24. Xiang, S. R., Cook, M., Saucier, S., Gillespie, P., Socha, R., Scroggins, R., et al. (2010). Development of amplified fragment length polymorphism-derived functional strain-specific markers to assess the persistence of 10 bacterial strains in soil microcosms. Applied and Environmental Microbiology, 76, 7126–7135.

    Article  CAS  Google Scholar 

  25. Elizaquível, P., Aznar, R., & Sánchez, G. (2014). Recent developments in the use of viability dyes and quantitative PCR in the food microbiology field. Journal of Applied Microbiology, 116, 1–13.

    Article  CAS  Google Scholar 

  26. Daranas, N., Bonaterra, A., Francés, J., Cabrefiga, J., Montesinos, E., & Badosa, E. (2018). Monitoring viable cells of the biological control agent Lactobacillus plantarum PM411 in aerial plant surfaces by means of a strain-specific viability quantitative PCR method. Applied and Environmental Microbiology, 84, e00107–e118.

    Article  CAS  Google Scholar 

  27. Nocker, A., Cheung, C. Y., & Camper, A. K. (2006). Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. Journal of Microbiological Methods, 67, 310–320.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We acknowledge professor Zhi-yuan Chen and Teoh Peik Lin for improving this manuscript. This work was supported by funds from Heilongjiang Academy of Sciences for the Promotion of Basic Application Technology of Institute, Heilongjiang Province for the Applied Technology Research and Sustainable Development (GA19B105) and Natural Science Foundation of Heilongjiang (C2017059).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jing Li.

Additional information

Publisher's Note

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

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, S., Ma, Y., Jiang, W. et al. Development of a Strain-Specific Quantification Method for Monitoring Bacillus amyloliquefaciens TF28 in the Rhizospheric Soil of Soybean. Mol Biotechnol 62, 521–533 (2020). https://doi.org/10.1007/s12033-020-00268-6

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-020-00268-6

Keywords

Navigation