Journal of General Plant Pathology

, Volume 85, Issue 2, pp 142–154 | Cite as

Enhanced biocontrol of tomato bacterial wilt using the combined application of Mitsuaria sp. TWR114 and nonpathogenic Ralstonia sp. TCR112

  • Malek Marian
  • Akio Morita
  • Hiroyuki Koyama
  • Haruhisa Suga
  • Masafumi ShimizuEmail author
Disease Control


We previously identified Mitsuaria sp. TWR114 and nonpathogenic Ralstonia sp. TCR112 as potential biocontrol agents to suppress tomato bacterial wilt caused by Ralstonia pseudosolanacearum. Because commercial biocontrol products require a practical cost-effective application method that maximizes their performance, we investigated whether the combined application of TWR114 and TCR112 enhances the biocontrol of bacterial wilt. In pot experiments, all the tested inoculum ratios (i.e., 1:1, 1:2, and 2:1) of the TWR114 + TCR112 treatment significantly suppressed the incidence of bacterial wilt, even at 28 days post-challenge inoculation (dpi) (13–47% wilt incidence), while 60% of plants treated with the individual isolates developed bacterial wilt within 10–12 dpi. The pathogen population in the rhizosphere and aboveground regions decreased considerably after the TWR114 + TCR112 treatment compared with that in the individual treatments. Moreover, the pathogen population in the aboveground parts of TWR114 + TCR112-treated plants had decreased to an undetectable level by 28 dpi. After inoculation with the pathogen, the expression of several tomato defense-related genes was higher in the TWR114 + TCR112-treated plants than in those treated with the individual isolates. Altogether, the results indicate that TWR114 and TCR112 applied together have a synergistic suppressive effect and that stronger defense priming might contribute to the improved biocontrol. The combination of both isolates may be a very promising approach for controlling tomato bacterial wilt in the future.


Biological control Combined application Induced systemic resistance Priming Ralstonia pseudosolanacearum Synergistic effect 



This work was financially supported by JSPS KAKENHI (Grant Numbers JP24780317 and JP15KT0029).


  1. Ahn IP, Lee SW, Kim MG, Park SR, Hwang DJ, Bae SC (2011) Priming by rhizobacterium protects tomato plants from biotrophic and necrotrophic pathogen infections through multiple defense mechanisms. Mol Cells 32:7–14CrossRefPubMedPubMedCentralGoogle Scholar
  2. Aimé S, Alabouvette C, Steinberg C, Olivain C (2013) The endophytic strain Fusarium oxysporum Fo47: a good candidate for priming the defense responses in tomato roots. Mol Plant-Microbe Interact 26:918–926CrossRefPubMedGoogle Scholar
  3. Alizadeh H, Behboudi K, Ahmadzadeh M, Javan-Nikkhah M, Zamioudis C, Pieterse CMJ, Bakker PAHM (2013) Induced systemic resistance in cucumber and Arabidopsis thaliana by the combination of Trichoderma harzianum Tr6 and Pseudomonas sp. Ps14. Biol Control 65:14–23CrossRefGoogle Scholar
  4. Álvarez B, Biosca EG, López MM (2010) On the life of Ralstonia solanacearum, a destructive bacterial plant pathogen. In: Mendez-Vilas A (ed) Current research, technology and education topics in applied microbiology and microbial biotechnology. Formatex, Badajoz, pp 267–279Google Scholar
  5. Bardas GA, Lagopodi AL, Kadoglidou K, Tzavella-Klonari K (2009) Biological control of three Colletotrichum lindemuthianum races using Pseudomonas chlororaphis PCL1391 and Pseudomonas fluorescens WCS365. Biol Control 49:139–145CrossRefGoogle Scholar
  6. Boukaew S, Chuenchit S, Petcharat V (2011) Evaluation of Streptomyces spp. for biological control of Sclerotium root and stem rot and Ralstonia wilt of chili pepper. BioControl 56:365–374CrossRefGoogle Scholar
  7. Chen YY, Lin YM, Chao TC, Wang JF, Liu AC, Ho FI, Cheng CP (2009) Virus-induced gene silencing reveals the involvement of ethylene-, salicylic acid- and mitogen-activated protein kinase-related defense pathways in the resistance of tomato to bacterial wilt. Physiol Plantarum 136:324–335CrossRefGoogle Scholar
  8. Chen D, Liu X, Li C, Tian W, Shen Q, Shen B (2014) Isolation of Bacillus amyloliquefaciens S20 and its application in control of eggplant bacterial wilt. J Environ Manage 137:120–127CrossRefPubMedGoogle Scholar
  9. Domenech J, Reddy MS, Kloepper JW, Ramos B, Gutierrez-Manero J (2006) Combined application of the biological product LS213 with Bacillus, Pseudomonas or Chryseobacterium for growth promotion and biological control of soil-borne diseases in pepper and tomato. Biocontrol 51:245–258CrossRefGoogle Scholar
  10. Dunne C, Moënne-Loccoz Y, McCarthy J, Higgins P, Powell J, Dowling DN, O’Gara F (1998) Combining proteolytic and phloroglucinol-producing bacteria for improved biocontrol of Pythium-mediated damping-off of sugar beet. Plant Pathol 47:299–307CrossRefGoogle Scholar
  11. Elphinstone JG (2005) The current bacterial wilt situation: a global overview. In: Allen C, Piror P, Hayward AC (eds) Bacterial wilt disease and the Ralstonia solanacearum. complex. APS Press, St. Paul, pp 9–28Google Scholar
  12. Feng DX, Tasset C, Hanemian M, Barlet X, Hu J, Trémousaygue D, Deslandes L, Marco Y (2012) Biological control of bacterial wilt in Arabidopsis thaliana involves abscissic acid signalling. New Phytol 194:1035–1045CrossRefPubMedGoogle Scholar
  13. French ER, Gutarra L, Aley P, Elphinstone J (1995) Culture media for Pseudomonas solanacearum isolation, identification and maintenance. Fitopatologia 30:126–130Google Scholar
  14. Ghareeb H, Bozsó Z, Ott PG, Repenning C, Stahl F, Wydra K (2011) Transcriptome of silicon-induced resistance against Ralstonia solanacearum in the silicon non-accumulator tomato implicates priming effect. Physiol Mol Plant Pathol 75:83–89CrossRefGoogle Scholar
  15. Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu Rev Phytopathol 43:205–227CrossRefPubMedGoogle Scholar
  16. Harvás A, Landa B, Jiménez-Díaz RM (1997) Influence of chickpea genotype and Bacillus sp. on protection from Fusarium wilt by seed treatment with nonpathogenic Fusarium oxysporum. Eur J Plant Pathol 103:631–642CrossRefGoogle Scholar
  17. Hase S, Shimizu A, Nakaho K, Takenaka S, Takahashi H (2006) Induction of transient ethylene and reduction in severity of tomato bacterial wilt by Pythium oligandrum. Plant Pathol 55:537–543CrossRefGoogle Scholar
  18. Hase S, Takahashi S, Takenaka S, Nakaho K, Arie T, Seo S, Ohashi Y, Takahashi H (2008) Involvement of jasmonic acid signalling in bacterial wilt disease resistance induced by biocontrol agent Pythium oligandrum in tomato. Plant Pathol 57:870–876CrossRefGoogle Scholar
  19. Hendrick CA, Sequeira L (1984) Lipopolysaccharide-defective mutants of the wilt pathogen Pseudomonas solanacearum. Appl Environ Microbiol 48:94–101PubMedPubMedCentralGoogle Scholar
  20. Jetiyanon K (2007) Defensive-related enzyme response in plants treated with a mixture of Bacillus strains (IN937a and IN937b) against different pathogens. Biol Control 42:178–185CrossRefGoogle Scholar
  21. Jetiyanon K, Kloepper JW (2002) Mixtures of plant growth-promoting rhizobacteria for induction of systemic resistance against multiple plant diseases. Biol Control 24:285–291CrossRefGoogle Scholar
  22. Jetiyanon K, Fowler WD, Kloepper JW (2003) Broad-spectrum protection against several pathogens by PGPR mixtures under field conditions in Thailand. Plant Dis 87:1390–1394CrossRefGoogle Scholar
  23. Jogaiah S, Abdelrahman M, Tran LS, Shin-ichi I (2013) Characterization of rhizosphere fungi that mediate resistance in tomato against bacterial wilt disease. J Exp Bot 64:3829–3842CrossRefPubMedGoogle Scholar
  24. Liu Y, Shi J, Feng Y, Yang X, Li X, Shen Q (2013) Tobacco bacterial wilt can be biologically controlled by the application of antagonistic strains in combination with organic fertilizer. Biol Fertil Soils 49:447–464CrossRefGoogle Scholar
  25. Liu HX, Li SM, Luo YM, Luo LX, Li JQ, Guo JH (2014) Biological control of Ralstonia wilt, Phytophthora blight, Meloidogyne root-knot on bell pepper by the combination of Bacillus subtilis AR12, Bacillus subtilis SM21 and Chryseobacterium sp. R89. Eur J Plant Pathol 139:107–116CrossRefGoogle Scholar
  26. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2–∆∆CT method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  27. Lucas JA, Solano BR, Montes F, Ojeda J, Megias M, Mañero FG (2009) Use of two PGPR strains in the integrated management of blast disease in rice (Oryza sativa) in southern Spain. Field Crop Res 114:404–410CrossRefGoogle Scholar
  28. Mansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M, Ronald P, Dow M, Verdier V, Beer SV, Machado MA, Toth I, Salmond G, Foster GD (2012) Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol 13:614–629CrossRefPubMedGoogle Scholar
  29. Marian M, Nishioka T, Koyama H, Suga H, Shimizu M (2018) Biocontrol potential of Ralstonia sp. TCR112 and Mitsuaria sp. TWR114 against tomato bacterial wilt. Appl Soil Ecol 128:71–80CrossRefGoogle Scholar
  30. Martínez-Medina A, Fernández I, Sánchez-Guzmán MJ, Jung SC, Pascual JA, Pozo MJ (2013) Deciphering the hormonal signalling network behind the systemic resistance induced by Trichoderma harzianum in tomato. Front Plant Sci 4:206CrossRefPubMedPubMedCentralGoogle Scholar
  31. Milling A, Babujee L, Allen C (2011) Ralstonia solanacearum extracellular polysaccharide is a specific elicitor of defense responses in wilt-resistant tomato plants. PLoS ONE 6:e15853CrossRefPubMedPubMedCentralGoogle Scholar
  32. Myresiotis CK, Karaoglanidis GS, Vryzas Z, Papadopoulou-Mourkidou E (2012) Evaluation of plant-growth-promoting rhizobacteria, acibenzolar-S-methyl and hymexazol for integrated control of Fusarium crown and root rot on tomato. Pest Manag Sci 68:404–411CrossRefPubMedGoogle Scholar
  33. Niu DD, Liu HX, Jiang CH, Wang YP, Wang QY, Jin HL, Guo JH (2011) The plant growth-promoting rhizobacterium Bacillus cereus AR156 induces systemic resistance in Arabidopsis thaliana by simultaneously activating salicylate- and jasmonate/ethylene-dependent signaling pathways. Mol Plant-Microbe Interact 24:533–542CrossRefPubMedGoogle Scholar
  34. Niu DD, Wang CJ, Guo YH, Jiang CH, Zhang WZ, Wang YP, Guo JH (2012) The plant growth-promoting rhizobacterium Bacillus cereus AR156 induces resistance in tomato with induction and priming of defence response. Biocontrol Sci Technol 22:991–1004CrossRefGoogle Scholar
  35. Pierson EA, Weller DM (1994) Use of mixtures of fluorescent pseudomonads to suppress take-all and improve the growth of wheat. Phytopathology 84:940–947CrossRefGoogle Scholar
  36. Pieterse CM, Leon-Reyes A, Van der Ent S, Van Wees SC (2009) Networking by small-molecule hormones in plant immunity. Nat Chem Biol 5:308–316CrossRefPubMedGoogle Scholar
  37. Raupach GS, Kloepper JW (1998) Mixtures of plant growth-promoting rhizobacteria enhance biological control of multiple cucumber pathogens. Phytopathology 88:1158–1164CrossRefPubMedGoogle Scholar
  38. Roberts DP, Lohrke SM, Meyer SL, Buyer JS, Bowers JH, Baker CJ, Li W, de Souza JT, Lewis JA, Chung S (2005) Biocontrol agents applied individually and in combination for suppression of soilborne diseases of cucumber. Crop Protect 24:141–155CrossRefGoogle Scholar
  39. Safni I, Cleenwerck I, De Vos P, Fegan M, Sly L, Kappler U (2014) Polyphasic taxonomic revision of the Ralstonia solanacearum species complex: proposal to emend the descriptions of Ralstonia solanacearum and Ralstonia syzygii and reclassify current R. syzygii strains as Ralstonia syzygii subsp. syzygii subsp. nov., R. solanacearum phylotype IV strains as Ralstonia syzygii subsp. indonesiensis subsp. nov., banana blood disease bacterium strains as Ralstonia syzygii subsp. celebesensis subsp. nov. and R. solanacearum phylotype I and III strains as Ralstonia pseudosolanacearum. Int J Syst Evol Microbiol 64:3087–3103CrossRefPubMedGoogle Scholar
  40. Santiago CD, Yagi S, Ijima M, Nashimoto T, Sawada M, Ikeda S, Asano K, Orikasa Y, Ohwada T (2017) Bacterial compatibility in combined inoculations enhances the growth of potato seedlings. Microbes Environ 32:14–23CrossRefPubMedPubMedCentralGoogle Scholar
  41. Sarma BK, Yadav SK, Singh S, Singh HB (2015) Microbial consortium-mediated plant defense against phytopathogens: readdressing for enhancing efficacy. Soil Biol Biochem 87:25–33CrossRefGoogle Scholar
  42. Singh PP, Shin YC, Park CS, Chung YR (1999) Biological control of Fusarium wilt of cucumber by chitinolytic bacteria. Phytopathology 89:92–99CrossRefPubMedGoogle Scholar
  43. Singh R, Soni SK, Kalra A (2013) Synergy between Glomus fasciculatum and a beneficial Pseudomonas in reducing root diseases and improving yield and forskolin content in Coleus forskohlii Briq. under organic field conditions. Mycorrhiza 23:35–44CrossRefPubMedGoogle Scholar
  44. Spadaro D, Gullino ML (2005) Improving the efficacy of biocontrol agents against soilborne pathogens. Crop Protect 24:601–613CrossRefGoogle Scholar
  45. Srivastava R, Khalid A, Singh US, Sharma AK (2010) Evaluation of arbuscular mycorrhizal fungus, fluorescent Pseudomonas and Trichoderma harzianum formulation against Fusarium oxysporum f. sp. lycopersici for the management of tomato wilt. Biol Control 53:24–31CrossRefGoogle Scholar
  46. Sundaramoorthy S, Raguchander T, Ragupathi N, Samiyappan R (2012) Combinatorial effect of endophytic and plant growth promoting rhizobacteria against wilt disease of Capsicum annum L. caused by Fusarium solani. Biol Control 60:59–67Google Scholar
  47. Suzuki Y, Hibino T, Kawazu T, Wada T, Kihara T, Koyama H (2003) Extraction of total RNA from leaves of Eucalyptus and other woody and herbaceous plants using sodium isoascorbate. Biotechniques 34:988–993CrossRefPubMedGoogle Scholar
  48. Takahashi H, Nakaho K, Ishihara T, Ando S, Wada T, Kanayama Y, Asano S, Yoshida S, Tsushima S, Hyakumachi M (2014) Transcriptional profile of tomato roots exhibiting Bacillus thuringiensis-induced resistance to Ralstonia solanacearum. Plant Cell Rep 33:99–110CrossRefPubMedGoogle Scholar
  49. Wu K, Su L, Fang Z, Yuan S, Wang L, Shen B, Shen Q (2017) Competitive use of root exudates by Bacillus amyloliquefaciens with Ralstonia solanacearum decreases the pathogenic population density and effectively controls tomato bacterial wilt. Sci Hort 218:132–138CrossRefGoogle Scholar
  50. Xu XM, Jeffries P, Pautasso M, Jeger MJ (2011) Combined use of biocontrol agents to manage plant diseases in theory and practice. Phytopathology 101:1024–1031CrossRefPubMedGoogle Scholar
  51. Xue QY, Chen Y, Li SM, Chen LF, Ding GC, Guo DW, Guo JH (2009) Evaluation of the strains of Acinetobacter and Enterobacter as potential biocontrol agents against Ralstonia wilt of tomato. Biol Control 48:252–258CrossRefGoogle Scholar
  52. Yabuuchi E, Kosako Y, Yano I, Hotta H, Nishiuchi Y (1995) Transfer of two Burkholderia and an Alcaligenes species to Ralstonia gen. nov.: proposal of Ralstonia pickettii (Ralston, Palleroni and Doudoroff 1973) comb. nov., Ralstonia solanacearum (Smith 1896) comb. nov. and Ralstonia eutropha (Davis 1969) comb. nov. Microbiol Immunol 39:897–904CrossRefPubMedGoogle Scholar
  53. Yamamoto S, Shiraishi S, Kawagoe Y, Mochizuki M, Suzuki S (2015) Impact of Bacillus amyloliquefaciens S13-3 on control of bacterial wilt and powdery mildew in tomato. Pest Manag Sci 71:722–727CrossRefPubMedGoogle Scholar
  54. Yuan S, Li M, Fang Z, Liu Y, Shi W, Pan B, Wu K, Shi J, Shen B, Shen Q (2016) Biological control of tobacco bacterial wilt using Trichoderma harzianum amended bioorganic fertilizer and the arbuscular mycorrhizal fungi Glomus mosseae. Biol control 92:164–171CrossRefGoogle Scholar
  55. Yuliar NA, Toyota K (2015) Recent trends in control methods for bacterial wilt diseases caused by Ralstonia solanacearum. Microbes Environ 30:1–11CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© The Phytopathological Society of Japan and Springer Japan KK, part of Springer Nature 2019

Authors and Affiliations

  1. 1.The United Graduate School of Agricultural ScienceGifu UniversityGifuJapan
  2. 2.Faculty of AgricultureShizuoka UniversityShizuokaJapan
  3. 3.Life Science Research CenterGifu UniversityGifuJapan

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