Skip to main content
SpringerLink
Log in

Bacillus amyloliquefaciens T-5 may prevent Ralstonia solanacearum infection through competitive exclusion

  • Original Paper
  • Published:
Biology and Fertility of Soils Aims and scope Submit manuscript

Abstract

Investigation of the properties and mechanisms of the interactions of root-colonizing biocontrol bacteria and plant pathogens is necessary to optimize the biocontrol strategies. In the present study, the interaction of a biocontrol strain Bacillus amyloliquefaciens T-5 tagged with a green fluorescent protein marker and a bacterial wilt pathogen Ralstonia solanacearum QL-Rs1115 tagged with red fluorescent protein marker was studied on tomato roots using different inoculation methods. The results showed that in the co-culture experiment, the population of pathogen QL-RFP was decreased by increasing the initial inoculum concentration of biocontrol strain. In the greenhouse experiment, both strains T-5-GFP and QL-RFP colonized tomato roots (root tips, root hairs, primary roots, and root junctions) and formed a biofilm on the root surfaces as determined by dilution plating and confocal laser scanning microscopy (CLSM) techniques. However, the root colonization of pathogen strain QL-RFP was almost completely suppressed in the presence of biocontrol strain T-5-GFP when both soil and plant seedlings were treated with T-5-GFP. The results of this study revealed the effectiveness of strain B. amyloliquefaciens T-5 as a biocontrol agent against tomato wilt pathogen and the significance of inoculation method used to inoculate biocontrol strain.

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
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Al-Waili N, Salom K, Al-Ghamdi A, Ansari MJ (2012) Antibiotic, pesticide, and microbial contaminants of honey: human health hazards. Scie World J 2012:930849

    Google Scholar 

  • Aliye N, Fininsa C, Hiskias Y (2008) Evaluation of rhizosphere bacterial antagonists for their potential to bioprotect potato (Solanum tuberosum) against bacterial wilt (Ralstonia solanacearum). Biol Control 47:282–288

    Article  Google Scholar 

  • Bais HP, Fall R, Vivanco JM (2004) Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production. Plant Physiol 134:307–319

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bhattacharyya PN, Jha DK (2012) Plant growth-promoting rhizobacteria (PGPR): emergence in agriculture. World J Microbiol Biotechnol 28:1327–1350

    Article  CAS  PubMed  Google Scholar 

  • Cao Y, Zhang Z, Ling N, Yuan Y, Zheng X, Shen B, Shen Q (2011) Bacillus subtilis SQR 9 can control Fusarium wilt in cucumber by colonizing plant roots. Biol Fert Soils 47:495–506

    Article  CAS  Google Scholar 

  • Compant S, Clément C, Sessitsch A (2010) Plant growth-promoting bacteria in the rhizo- and endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization. Soil Biol Biochem 42:669–678

    Article  CAS  Google Scholar 

  • Czajkowski R, de Boer W, van Veen J, van der Wolf J (2012) Studies on the interaction between the biocontrol agent, Serratia plymuthica A30, and blackleg-causing Dickeya sp. (biovar 3) in potato (Solanum tuberosum). Plant Pathol 61:677–688

    Article  Google Scholar 

  • D’Alvise PW, Magdenoska O, Melchiorsen J, Nielsen KF, Gram L (2014) Biofilm formation and antibiotic production in Ruegeria mobilis are influenced by intracellular concentrations of cyclic dimeric guanosinmonophosphate. Environ Microbiol 16:1252–1266

    Article  PubMed  Google Scholar 

  • Elphinstone J, Hennessy J, Wilson J, Stead D (1996) Sensitivity of different methods for the detection of Ralstonia solanacearum in potato tuber extracts. EPPO Bull 26:663–678

    Article  Google Scholar 

  • Errampalli D, Leung K, Cassidy M, Kostrzynska M, Blears M, Lee H, Trevors J (1999) Applications of the green fluorescent protein as a molecular marker in environmental microorganisms. J Microbiol Meth 35:187–199

    Article  CAS  Google Scholar 

  • Falk P, Borén T, Haslam D, Caparon M (1995) Bacterial adhesion and colonization assays. Method Cell Biol 45:165–192

    Article  Google Scholar 

  • Fan B, Chen XH, Budiharjo A, Bleiss W, Vater J, Borriss R (2011) Efficient colonization of plant roots by the plant growth promoting bacterium Bacillus amyloliquefaciens FZB42, engineered to express green fluorescent protein. J Biotechnol 151:303–311

    Article  CAS  PubMed  Google Scholar 

  • Goedhart J, Van Weeren L, Hink MA, Vischer NO, Jalink K, Gadella TW (2010) Bright cyan fluorescent protein variants identified by fluorescence lifetime screening. Nat Methods 7:137–139

    Article  CAS  PubMed  Google Scholar 

  • Hayward AC (1991) Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Annu Rev Phytopathol 29:65–87

    Article  CAS  PubMed  Google Scholar 

  • Heim R, Tsien RY (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6:178–182

    Article  CAS  PubMed  Google Scholar 

  • Huang Q, Yan X, Wang JF (2012) Improved biovar test for Ralstonia solanacearum. J Microbiol Meth 88:271–274

    Article  CAS  Google Scholar 

  • Janse JD (1996) Potato brown rot in western Europe. History, present occurrence and some remarks on possible origin, epidemiology and control strategies. Bull OEPP 26:679–695

    Article  Google Scholar 

  • Kamilova F, Validov S, Azarova T, Mulders I, Lugtenberg B (2005) Enrichment for enhanced competitive plant root tip colonizers selects for a new class of biocontrol bacteria. Environ Microbiol 7:1809–1817

    Article  CAS  PubMed  Google Scholar 

  • Kato A, Miyake T, Nishigata K, Tateishi H, Teraoka T, Arie T (2012) Use of fluorescent proteins to visualize interactions between the Bakanae disease pathogen Gibberella fujikuroi and the biocontrol agent Talaromyces sp. KNB-422. J Gen Plant Pathol 78:54–61

    Article  CAS  Google Scholar 

  • Kempe J, Sequeira L (1983) Biological control of bacterial wilt of potatoes: attempts to induce resistance by treating tubers with bacteria. Plant Dis 67:499–503

    Article  Google Scholar 

  • Kurabachew H, Stahl F, Wydra K (2013) Global gene expression of rhizobacteria-silicon mediated induced systemic resistance in tomato (Solanum lycopersicum) against Ralstonia solanacearum. Physiol Mol Plant P 84:44–52

    Article  CAS  Google Scholar 

  • Ling N, Zhang W, Tan S, Huang Q, Shen Q (2012) Effect of the nursery application of bioorganic fertilizer on spatial distribution of Fusarium oxysporum f. sp. niveum and its antagonistic bacterium in the rhizosphere of watermelon. Appl Soil Ecol 59:13–19

    Article  Google Scholar 

  • Masunaka A, Nakaho K, Sakai M, Takahashi H, Takenaka S (2009) Visualization of Ralstonia solanacearum cells during biocontrol of bacterial wilt disease in tomato with Pythium oligandrum. J Gen Plant Pathol 75:281–287

    Article  Google Scholar 

  • Nihorimbere V, Cawoy H, Seyer A, Brunelle A, Thonart P, Ongena M (2012) Impact of rhizosphere factors on cyclic lipopeptide signature from the plant beneficial strain Bacillus amyloliquefaciens S499. FEMS Microbiol Ecol 79:176–191

    Article  CAS  PubMed  Google Scholar 

  • Njoloma J, Tanaka K, Shimizu T, Nishiguchi T, Zakria M, Akashi R, Oota M, Akao S (2006) Infection and colonization of aseptically micropropagated sugarcane seedlings by nitrogen-fixing endophytic bacterium, Herbaspirillum sp. B501gfp1. Biol Fert Soils 43:137–143

    Article  Google Scholar 

  • Oliveira PH, Prather KJ, Prazeres DM, Monteiro GA (2009) Structural instability of plasmid biopharmaceuticals: challenges and implications. Trends Biotechnol 27:503–511

    Article  CAS  PubMed  Google Scholar 

  • Palaniyandi SA, Yang SH, Zhang L, Suh J-W (2013) Effects of actinobacteria on plant disease suppression and growth promotion. Appl Microbiol Biotechnol 97:9621–9636

    Article  CAS  PubMed  Google Scholar 

  • Qiu M, Li S, Zhou X, Cui X, Vivanco JM, Zhang N, Shen Q, Zhang R (2014) De-coupling of root-microbiome associations followed by antagonist inoculation improves rhizosphere soil suppressiveness. Biol Fert Soils 50:217–224

    Article  CAS  Google Scholar 

  • Ramesh R, Phadke GS (2012) Rhizosphere and endophytic bacteria for the suppression of eggplant wilt caused by Ralstonia solanacearum. Crop Prot 37:35–41

    Article  Google Scholar 

  • Raza W, Faheem M, Yousaf S, Rajer FU, Yameen M (2013) Volatile and non-volatile antifungal compounds produced by Trichoderma harzianum SQR-T037 suppressed the growth of Fusarium oxysporum f. sp. niveum. Sci Lett 1:21–24

    Google Scholar 

  • Ren X, Zhang N, Cao M, Wu K, Shen Q, Huang Q (2012) Biological control of tobacco black shank and colonization of tobacco roots by a Paenibacillus polymyxa strain C5. Biol Fert Soils 48:613–620

    Article  Google Scholar 

  • Rochat L, Péchy-Tarr M, Baehler E, Maurhofer M, Keel C (2010) Combination of fluorescent reporters for simultaneous monitoring of root colonization and antifungal gene expression by a biocontrol Pseudomonad on cereals with flow cytometry. Mol Plant Microbe In 23:949–961

    Article  CAS  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  • Schoina C, Stringlis IA, Pantelides IS, Tjamos SE, Paplomatas EJ (2011) Evaluation of application methods and biocontrol efficacy of Paenibacillus alvei strain K-165, against the cotton black root rot pathogen Thielaviopsis basicola. Biol Control 58:68–73

    Article  Google Scholar 

  • Swanson JK, Yao J, Tans-Kersten J, Allen C (2005) Behavior of Ralstonia solanacearum race 3 biovar 2 during latent and active infection of geranium. Phytopathol 95:136–143

    Article  Google Scholar 

  • Takenaka S, Sekiguchi H, Nakaho K, Tojo M, Masunaka A, Takahashi H (2008) Colonization of Pythium oligandrum in the tomato rhizosphere for biological control of bacterial wilt disease analyzed by real-time PCR and confocal laser-scanning microscopy. Phytopathol 98:187–195

    Article  CAS  Google Scholar 

  • Tan S, Dong Y, Liao H, Huang J, Song S, Xu Y, Shen Q (2013a) Antagonistic bacterium Bacillus amyloliquefaciens induces resistance and controls the bacterial wilt of tomato. Pest Manag Sci 69:1245–1252

    CAS  PubMed  Google Scholar 

  • Tan S, Jiang Y, Song S, Huang J, Ling N, Xu Y, Shen Q (2013b) Two Bacillus amyloliquefaciens strains isolated using the competitive tomato root enrichment method and their effects on suppressing Ralstonia solanacearum and promoting tomato plant growth. Crop Prot 43:134–140

    Article  Google Scholar 

  • Tans-Kersten J, Brown D, Allen C (2004) Swimming motility, a virulence trait of Ralstonia solanacearum, is regulated by FlhDC and the plant host environment. Mol Plant Microbe In 17:686–695

    Article  CAS  Google Scholar 

  • Timmusk S, Grantcharova N, Wagner EG (2005) Paenibacillus polymyxa invades plant roots and forms biofilms. Appl Environ Microbiol 71:7292–7300

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Torres AR, Araújo WL, Cursino L, de Barros RP, Mondin M, Hungria M, Azevedo JL (2013) Colonization of Madagascar periwinkle (Catharanthus roseus), by endophytes encoding gfp marker. Arch Microbiol 195:483–489

    Article  CAS  PubMed  Google Scholar 

  • Turner J, Backman P (1991) Factors relating to peanut yield increases after seed treatment with Bacillus subtilis. Plant Dis 75:347–353

    Article  Google Scholar 

  • Underwood W, Koh S, Somerville SC (2011) Visualizing cellular dynamics in plant-microbe interactions using fluorescent-tagged proteins. Methods Mol Biol 712:283–291

    Article  CAS  PubMed  Google Scholar 

  • Unge A, Tombolini R, Mølbak L, Jansson JK (1999) Simultaneous monitoring of cell number and metabolic activity of specific bacterial populations with a dual gfp-luxAB marker system. Appl Environ Microb 65:813–821

    CAS  Google Scholar 

  • Vasse J, Frey P, Trigalet A (1995) Microscopic studies of intercellular infection and protoxylem invasion of tomato roots by Pseudomonas solanacearum. Mol Plant Microbe In 8:241–251

    Article  CAS  Google Scholar 

  • Wang P, Zhong Z, Zhou J, Cai T, Zhu J (2008) Exopolysaccharide biosynthesis is important for Mesorhizobium tianshanense: plant host interaction. Arch Microbiol 189:525–530

    Article  CAS  PubMed  Google Scholar 

  • Wei Z, Yang X, Yin S, Shen Q, Ran W, Xu Y (2011) Efficacy of Bacillus-fortified organic fertiliser in controlling bacterial wilt of tomato in the field. Appl Soil Ecol 48:152–159

    Article  Google Scholar 

  • Xue Q, Chen Y, Li S, Chen L, Ding G, Guo D, Guo J (2009) Evaluation of the strains of Acinetobacter and Enterobacter as potential biocontrol agents against Ralstonia wilt of tomato. Biol Control 48:252–258

    Article  Google Scholar 

  • Yu X, Ai C, Xin L, Zhou G (2011) The siderophore-producing bacterium, Bacillus subtilis CAS15, has a biocontrol effect on Fusarium wilt and promotes the growth of pepper. Eur J Soil Biol 47:138–145

    Article  Google Scholar 

  • Zhang N, Wu K, He X, Li S, Zhang Z, Shen B, Yang X, Zhang R, Huang Q, Shen Q (2011) A new bioorganic fertilizer can effectively control banana wilt by strong colonization with Bacillus subtilis N11. Plant Soil 344:87–97

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This research was financially supported by the National Natural Science Foundation of China (41301262), the Innovative Research Team Development Plan of the Ministry of Education of China (IRT1256), the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and the 111 project (B12009). We would like to acknowledge Prof. Paolo Nannipieri from University of Florence, Italy, for his careful revisions of the manuscript.

Author information

Authors and Affiliations

  1. National Engineering Research Center for Organic-based Fertilizers, Nanjing Agricultural University, Nanjing, 210095, China

    Shiyong Tan, Yian Gu, Chunlan Yang, Yue Dong, Xinlan Mei, Qirong Shen & Yangchun Xu

  2. Key Laboratory of Plant Nutrition and Biological Fertilizer, Ministry of Agriculture, Changsha, 410205, Hunan, People’s Republic of China

    Shiyong Tan

  3. Jiangsu Collaborative Innovation Center for Solid Organic Waste Utilization, Nanjing Agricultural University, Nanjing, 210095, China

    Shiyong Tan, Yian Gu, Chunlan Yang, Yue Dong, Xinlan Mei, Qirong Shen & Yangchun Xu

  4. Hunan Taigu Biotechnology Co., Ltd, Changsha, 410205, Hunan, China

    Shiyong Tan

Authors
  1. Shiyong Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yian Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chunlan Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yue Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xinlan Mei
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Qirong Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yangchun Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yangchun Xu.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOCX 476 kb)

ESM 2

(DOCX 114 kb)

ESM 3

(DOCX 1366 kb)

ESM 4

(DOCX 1039 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tan, S., Gu, Y., Yang, C. et al. Bacillus amyloliquefaciens T-5 may prevent Ralstonia solanacearum infection through competitive exclusion. Biol Fertil Soils 52, 341–351 (2016). https://doi.org/10.1007/s00374-015-1079-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00374-015-1079-z

Keywords

Search

Navigation