Plant and Soil

, Volume 344, Issue 1–2, pp 87–97 | Cite as

A new bioorganic fertilizer can effectively control banana wilt by strong colonization with Bacillus subtilis N11

  • Nan Zhang
  • Kai Wu
  • Xin He
  • Shu-qing Li
  • Zhen-hua Zhang
  • Biao Shen
  • Xing-ming Yang
  • Rui-fu Zhang
  • Qi-wei Huang
  • Qi-rong Shen
Regular Article


Fusarium wilt is one of the most serious diseases caused by a soil-borne pathogen affecting banana production. The goal of this study was to evaluate the capability of a novel bio-organic fertilizer (BIO2) that integrated the biocontrol agent Bacillus subtilis N11, and mature composts to control Fusarium wilt of banana in pot experiments. The results showed that the application of the BIO2 significantly decreased the incidence rate of Fusarium wilt compared to the control. To determine the antagonistic mechanism of the strain, we also studied the colonization of the natural biocontrol agent on banana roots using a GFP marker. The studies were performed in a hydroponic culture system, a sand system and a natural soil system. The results indicated that the bacteria colonized predominantly by forming biofilms along the elongation and differentiation zones of the roots. The fact that similar observations were obtained in all three systems suggests that colonization by N11 can be studied in a defined system. The population of B. subtilis N11 in the rhizosphere and on banana roots was also monitored. We speculate that the colonization pattern of B.subtilis N11 can be linked to the mechanism of protection of plants from fungal infection.


Fusarium wilt Bio-organic fertilizer (BIO) Banana Bacillus subtilis Green fluorescent protein (GFP) Colonization 



Bioorganic fertilizer


Colony forming unit


Confocal laser scanning microscopy


Fusarium oxysporum f. sp. cubense


Green fluorescent protein



We gratefully acknowledge Agricultural Ministry of China (201103004), Nature Science Foundation of China (31070462) and Ministry of Science and Technology of China (2010AA10Z401).


  1. Anith KN, Manomohandas TP (2001) Combined application of Trichoderma harzianum, Alcaligenes sp. strain AMB 8 for controlling nursery rot disease of black pepper. Indian Phytopathol 54:335–339Google Scholar
  2. Assmus B, Hutzler P, Kirchhof G, Amann R, Lawrence JK, Hartmann A (1995) In situ localization of Azospirillum brasilense in the rhizosphere of wheat with fluorescently labeled rRNA-target oligonucleotide probes, scanning confocal laser microscopy. Appl Environ Microbiol 61:1013–1019PubMedGoogle Scholar
  3. Bais HP, Weir TL, Perry LG, Gilroy S, Vivanco JM (2006) The role of root exudates in rhizosphere interactions with plants, other organisms. Annu Rev Plant Biol 57:233–266PubMedCrossRefGoogle Scholar
  4. Bloemberg GV, Wijifjes AHM, Lamers GEM, Stuurman N, Lugtenberg BJJ (2000) Simultaneous imaging of Pseudomonas fluorescens WCS365 populations expressing three different autofluorescent Proteins in the Rhizosphere, New Perspectives for Studying Microbial Communities. Mol Plant Microb Interact 13:1170–1176CrossRefGoogle Scholar
  5. Brigidi P, De Rossi E, Riccard G, Matteuzzi D (1991) A highly efficient electroporation system for transformation of Bacillus licheniforms. Biotechnol Tech 5:5–8CrossRefGoogle Scholar
  6. Cavaglieri L, Orlando J, Rodriguez MI (2005) Biocontrol of Bacillus subtilis against Fusarium verticillioides in vitro, at the maize root level. Res Microbiol 156:748–754PubMedCrossRefGoogle Scholar
  7. Chalfie M, Euskirchen YTG, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 264:802–805CrossRefGoogle Scholar
  8. Chelius MK, Triplett EW (2000) Immunolocalization of dinitrogenase reductase produced by Klebsiela pneumoniae in association with Zea mays L. Appl Environ Microbiol 66:783–787PubMedCrossRefGoogle Scholar
  9. Chen LH, Yang XM, Raza W, Li JH, Zhang FG, Tang Z, Liu YX, Shen QR (2010) Trichoderma harzianum SQR-T037 rapidly degrades allelochemicals in rhizospheres of continuously cropped cucumbers. Appl Microbiol Technol (on line)Google Scholar
  10. Compant S, Duffy B, Nowak J, Clement C, Barka EA (2005) Use of plant growth-promoting bacteria for biocontrol of plant diseases principles, mechanisms of action, future prospects. Appl Environ Microbiol 71:2951–4959Google Scholar
  11. Compant S, Clement C, Sessitsch A (2010) Plant growth-promoting bacteria in the rhizo-, endosphere of plants: their role, colonization, mechanisms involved, prospects for utilization. Soil Biol Biochem 42:669–678CrossRefGoogle Scholar
  12. Cotxarrera L, Trillas-Gay MI, Steinberg C, Alabouvette C (2002) Use of sewage sludge compost, Trichoderma asperellum isolates to suppress Fusarium wilt of tomato. Soil Biol Biochem 34:467–476CrossRefGoogle Scholar
  13. Dandurand LM, Schotzko DJ, Knudsen GR (1997) Spatial patterns of rhizoplane populations of Pseudomonas fluorescens. Appl Environ Microbiol 63:3211–3217PubMedGoogle Scholar
  14. de Weger LA, Dekkers LC, van der Bij A, Lugtenberg BJJ (1993) Use of phosphate-reporter bacteria to study phosphate limitation in the rhizosphere, in bulk soil. Mol Plant Microb Interact 7:32–38CrossRefGoogle Scholar
  15. Duffy BK, Weller DM (1996) Biological control of take-all of wheat in the Pacific North-west of the USA using hypovirulent Gaeumannomyces graminis var. tritici, fluorescent Pseudomonads. J Phytopathol 144:11–12CrossRefGoogle Scholar
  16. Dunne C, Delany I, Fenton A, O’Gara F (1996) Mechanisms involved in biocontrol by microbial inoculants. Agronomie 16:721–729CrossRefGoogle Scholar
  17. Getha K, Vikineswary S (2002) Antagonistic effects of Streptomyces violaceusniger strain G10 on Fusarium oxysporum f. sp. cubense race 4: indirect evidence for the role of antibiosis in the antagonistic process. J Ind Microbiol Biotechnol 28:303–310PubMedCrossRefGoogle Scholar
  18. Getha K, Vikineswary S, Wong WH, Seki T, Ward A, Goodfellow M (2005) Evaluation of Streptomyces sp. strain g10 for suppression of Fusarium wilt, rhizosphere colonization in pot-grown banana plantlet. J Ind Microbiol Biotechnol 32:24–32PubMedCrossRefGoogle Scholar
  19. Gilroy S, Jones DL (2000) Through form to function, root hair development, nutrient uptake. Trends Plant Sci 5:56–60PubMedCrossRefGoogle Scholar
  20. Hiddink GA, van Bruggen AHC, Raaijmakers JM, Semrnoy AV (2005) Effect of organic management of soils on suppressiveness to Gaeumannomyces graminis var. tritici, its antagonist, Pseudomonas fluorescens. Eur J Plant Pathol 113:417–435CrossRefGoogle Scholar
  21. Jaeger CH, Lindow SE, Miller W, Clark E, Firestone MK (1999) Mapping of sugar, amino acid availability in soil around roots with bacterial sensors of sucrose, tryptophan. Appl Environ Microbiol 65:2685–2690PubMedGoogle Scholar
  22. Lin YH, Chang JY, Liu ET, Chao CP, Huang JW, Fang P, Chang LD (2009) Development of a molecular marker for specific detection of Fusarium oxysporum f. sp. cubense race 4. Eur J Plant Pathol 123:353–365CrossRefGoogle Scholar
  23. Ling N, Xue C, Huang QW, Yang XM, Xu YC, Shen QR (2010) Development of a mode of application of bio-organic fertilizer for improving the biocontrol efficacy to Fusarium wilt. Biocontrol 55:673–683CrossRefGoogle Scholar
  24. Liu XM, Zhou HX, Chen SF (2006) Colonization of Maize, Rice plants by strain Bacillus megaterium C4. Curr Microbiol 52:186–190PubMedCrossRefGoogle Scholar
  25. Luo J, Ran W, Hu J, Yang XM, Xu YC, Shen QR (2010) Application of bioorganic fertilizer significantly affected fungal diversity of soils. Soil Sci Soc Amer J 74:2039–2048Google Scholar
  26. Marschner P, Rengel Z (2007) Contributions of rhizosphere interactions to soil biological fertility. Soil Biological Fertility 81–98Google Scholar
  27. Mcinroy JA, Kloepper JW (1994) Studies on the indigenous endophytic bacteria of sweet corn, cotton. In: O’Gara F, Dowling DN, Boesten B (eds) Molecular ecology of rhizosphere micro-organisms. VCH Press, pp 19–28Google Scholar
  28. Njoloma J, Tanaka K, Shimizu T, Nishiguchi T, Zakria M, Akashi R, Oota M, Akao S (2006) Infection, colonization of aseptically micropropagated sugarcane seedlings by nitrogen-fixing endophytic bacterium, Herbaspirillum sp. B501gfp1. Biol Fertil Soils 43:137–143CrossRefGoogle Scholar
  29. O’Donnell K, Kistler HC, Cigelnik E, Ploetz RC (1998) Multiple evolutionary origins of the fungus causing Panama disease of banana: Concordant evidence from nuclear, mitochondrial gene genealogies. PNAS 95:2044–2049PubMedCrossRefGoogle Scholar
  30. Ploetz RC (1990) Population biology of Fusarium oxysporum f.sp.cubense. In: Ploetz RC (ed) Fusarium wilt of banana. APS, St. PaulGoogle Scholar
  31. Prosser JI (1994) Molecular marker systems for detection of genetically engineered micro-organisms in the environment. Microbiol 140:5–17CrossRefGoogle Scholar
  32. Raguchander T, Jayashree K, Samiyappan R (1997) Management of Fusarium wilt of banana using antagonistic microorganisms. J Biol Control 11:101–105Google Scholar
  33. Ramey BE, Koutsoudis M, von Bodman SB, Fuqua C (2004) Biofilm formation in plant-microbe associations. Curr Opin Microbiol 7:602–609PubMedCrossRefGoogle Scholar
  34. Ramos C, Molbak L, Molin S (2000) Bacterial activity in the rhizosphere analyzed at the single-cell level by monitoring ribosome contents, synthesis rates. Appl Environ Microbiol 66:801–809PubMedCrossRefGoogle Scholar
  35. Saravanan T, Muthusamy M, Marimuthu T (2003) Development of integrated approach to manage the fusarial wilt of banana. Crop Prot 22:1117–1123CrossRefGoogle Scholar
  36. Schonwitz R, Ziegler H (1989) Interaction of maize roots, rhizosphere microorganisms. J Plant Nutr Soil Sci 152:217–222CrossRefGoogle Scholar
  37. Sivamani E, Gnanamanickam SS (1988) Biological control of Fusarium oxysporum f.sp. cubense in banana by inoculation with Pseudomonas fluorescens. Plant Soil 107:3–9CrossRefGoogle Scholar
  38. Snyder W, Hanson H (1940) The species concept in Fusarium. Am J Bot 27:64–67CrossRefGoogle Scholar
  39. Timmusk S, Grantcharova N, Wagner EGH (2005) Paenibacillus polymyxa invades plant roots and forms biofilm. Appl Environ Microbiol 71:7292–7300PubMedCrossRefGoogle Scholar
  40. Tombolini R, van der Gaag DJ, Gerhardson B, Jansson JK (1999) Colonization pattern of the biocontrol strain Pseudomonas chlororaphis MA342 on barley seeds visualized by using green fluorescent protein. Appl Environ Microbiol 65:3674–3680PubMedGoogle Scholar
  41. Trevors JT, Chassy BM, Dower WJ, Blaschek HP (1992) Electrotransformation of bacteria by plasmid DNA. In: Chang DC, Chassy BM, Saunders JA, Sowers AE (eds) Guide to electroporation, electrofusion. Academic, San Diego, pp 265–290Google Scholar
  42. Trillas MI, Casanova E, Corxarrera L, Ordovas J, Borrero C, Aviles M (2006) Composts from agricultural waste, the Trichoderma asperellum strain T-34 suppress Rhizoctonia solani in cucumber seedlings. Biol Control 39:32–38CrossRefGoogle Scholar
  43. Unge A, Jansson JK (2001) Monitoring population size, activity, distribution of gfp-luxAB -tagged Pseudomonas fluorescens SBW25 during colonization of wheat. Microb Ecol 41:290–300PubMedGoogle Scholar
  44. von der Weid I, Artursson V, Seldin L, Jansson JK (2005) Antifungal, Root Surface Colonization Properties of GFP-Tagged Paenibacillus brasilensis PB177. World J Microbiol Biotechnol 12:1591–1597CrossRefGoogle Scholar
  45. Walker R, Rossall S, Ashe MJC (2002) Colonization of the developing rhizosphere of sugar beet seedlings by potential biocontrol agents applied as seed treatments. J Appl Microbiol 92:228–237PubMedCrossRefGoogle Scholar
  46. Walker TS, Bais HP, Grotewold E, Vivanco JM (2003) Root exudation, rhizosphere biology. Plant Physiol 132:44–51PubMedCrossRefGoogle Scholar
  47. Xue GP, Johnson JS, Dalrympl BP (1999) High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis, Bacillus licheniforms. J Microbiol Meth 34:183–191CrossRefGoogle Scholar
  48. Zhang SS, Raza W, Yang XM, Hu J, Huang QW, Xu YC, Liu XH, Ran W, Shen QR (2008) Control of Fusarium wilt disease of cucumber plants with the application of a bioorganic fertilizer. Biol Fertil Soils 44:1073–1080CrossRefGoogle Scholar
  49. Zhao QY, Dong CX, Yang XM, Mei XL, Ran W, Shen QR (2010) Biocontrol of Fusarium wilt disease for Cucumis melo melon using bio-organic fertilizer. Appl Soil Ecol 47:67–75Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Nan Zhang
    • 1
  • Kai Wu
    • 1
  • Xin He
    • 1
  • Shu-qing Li
    • 1
  • Zhen-hua Zhang
    • 1
  • Biao Shen
    • 1
  • Xing-ming Yang
    • 1
  • Rui-fu Zhang
    • 1
  • Qi-wei Huang
    • 1
  • Qi-rong Shen
    • 1
  1. 1.Jiangsu Key Laboratory for Solid Organic Waste UtilizationNanjing Agricultural UniversityNanjingChina

Personalised recommendations