Current Microbiology

, Volume 76, Issue 1, pp 100–107 | Cite as

In Vitro Formation of Dickeya zeae MS1 Biofilm

  • Ning Huang
  • Xiaoming Pu
  • Jingxin Zhang
  • Huifang Shen
  • Qiyun Yang
  • Zhongwen Wang
  • Birun LinEmail author


Bacterial soft rot caused by Dickeya zeae MS1 (Erwinia chrysanthemi) is one of the most devastating banana diseases worldwide. However, knowledge of the development and ecological interactions of D. zeae MS1 biofilm is limited. Here, we visualized the development and architecture of D. zeae MS1 biofilm using confocal laser scanning microscopy, and we evaluated the ability of D. zeae MS1 to form biofilms under different environmental conditions (carbon sources, temperatures, pH levels and mineral elements) using a microtiter plate assay. We found that the development of D. zeae MS1 biofilm could be categorized into four phases and that mature biofilm consisted of a highly organized architecture of both bacterial cells and a self-produced matrix of extracellular polysaccharides. Furthermore, sucrose was the most suitable carbon source for supporting the growth of biofilm cells and that 32 °C and pH 7.0 were the most favorable of the temperatures and pH levels examined. Meanwhile, the addition of Ca2+, Fe2+, K+ and Na+ enhanced the formation of biofilm in minimal medium cultures, whereas 2.5 mM Cu2+ and Mn2+ was inhibitory. A better understanding of biofilm formation under different environmental parameters will improve our knowledge of the growth kinetics of D. zeae MS1 biofilm.



We would like to thank Dr. Aiping Xu (Public Monitoring Center for Agro-product of Guangdong Academy of Agricultural Science, Guangdong, Guangzhou) and Weina Zhang (Centre for Agrobiological Gene Research, Guangdong Academy of Agricultural Sciences, Guangdong, Guangzhou) for their assistance with confocal laser scanning microscopy.


This work was supported by the Natural Science Foundation of Guangdong Province (No. 2015A030312002); the Guangzhou Science and Technology Project (No. 2014J4500034); the President Foundation of the Guangdong Academy of Agricultural Sciences (No. 201515).

Compliance with Ethical Standards

Conflict of interest

None declared.


  1. 1.
    Branda SS, Vik Å, Friedman L, Kolter R (2005) Biofilms: the matrix revisited. Trends Microbiol 13:20–26Google Scholar
  2. 2.
    Chavant P, Martinie B, Meylheuc T, Bellon-Fontaine MN, Hebraud M (2002) Listeria monocytogenes LO28: surface physicochemical properties and ability to form biofilms at different temperatures and growth phases. Appl Environ Microbiol 68:728–737Google Scholar
  3. 3.
    Cobine PA, Cruz LF, Navarrete F, Duncan D, Tygart M, de La Fuente L (2013) Xylella fastidiosa differentially accumulates mineral elements in biofilm and planktonic cells. PLoS ONE 8:e54936Google Scholar
  4. 4.
    Cruz LF, Cobine PA, de La Fuente L (2012) Calcium increases Xylella fastidiosa surface attachment, biofilm formation, and twitching motility. Appl Environ Microbiol 78:1321–1331Google Scholar
  5. 5.
    Czajkowski R, Grabe GJ, van der Wolf JM (2009) Distribution of Dickeya spp. and Pectobacterium carotovorum subsp. carotovorum in naturally infected seed potatoes. Eur J Plant Pathol 125:263–275Google Scholar
  6. 6.
    de Campos PA, Royer S, da Fonseca BDW, Araújo BF, Queiroz LL, de Brito CS, Gontijo-Filho PP, Ribas RM (2016) Multidrug resistance related to biofilm formation in Acinetobacter baumannii and Klebsiella pneumoniae clinical strains from different pulsotypes. Curr Microbiol 72(5):617–627Google Scholar
  7. 7.
    Elphinstone JG (2007) The canon of potato science: bacterial pathogens. Potato Res 50:247–249Google Scholar
  8. 8.
    Ghods S, Sims IM, Moradali MF, Rehm BHA (2015) Bactericidal compounds controlling growth of the plant pathogen Pseudomonas syringae pv. actinidiae, which forms biofilms composed of a novel exopolysaccharide. Appl Environ Microbiol 81(12):4026–4036Google Scholar
  9. 9.
    Hall-Stoodley L, Costerton JW, Stoodley P (2004) Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol 2:95–108Google Scholar
  10. 10.
    Hoffman LR, D’Argenio DA, MacCoss MJ, Zhang Z, Jones RA, Miller SI (2005) Aminoglycoside antibiotics induce bacterial biofilm formation. Nature 436:1171–1175Google Scholar
  11. 11.
    Jahn CE, Selimi DA, Barak JD, Charkowski A (2011) The Dickeya dadantii biofilm matrix consists of cellulose nanofibres, and is an emergent property dependent upon the type III secretion system and the cellulose synthesis operon. Microbiology 157:2733–2744Google Scholar
  12. 12.
    Jin Y, Samaranayake LP, Samaranayake Y, Yip HK (2004) Biofilm formation of Candida albicans is variably affected by saliva and dietary sugars. Arch Oral Biol 49:789–798Google Scholar
  13. 13.
    Kapsa JS (2008) Important threats in potato production and integrated pathogen/pest management. Potato Res 51:385–401Google Scholar
  14. 14.
    Karatan E, Watnick P (2009) Signals, regulatory networks, and materials that build and break bacterial biofilms. Microbiol Mol Biol Rev 73:310–347Google Scholar
  15. 15.
    Kieu NP, Aznar A, Segond D, Rigault M, Simond-Côte E, Kunz C, Soulay F, Expert D, Dellagi A (2012) Iron deficiency affects plant defence responses and confers resistance to Dickeya dadantii and Botrytis cinerea. Mol Plant Pathol 13:816–827Google Scholar
  16. 16.
    Killiny N, Martinez RH, Dumenyo CK, Cooksey DA, Almeida RPP (2013) The exopolysaccharide of Xylella fastidiosa is essential for biofilm formation, plant virulence, and vector transmission. Mol Plant Microbe Interact 26:1044–1053Google Scholar
  17. 17.
    Koczan JM, McGrath MJ, Zhao Y, Sundin GW (2009) Contribution of Erwinia amylovora exopolysaccharides amylovoran and levan to biofilm formation: implications in pathogenicity. Phytopathology 99:1237–1244Google Scholar
  18. 18.
    Laurila J, Hannukkala A, Nykyri J, Pasanen M, Hélias V, Garlant L, Pirhonen M (2010) Symptoms and yield reduction caused by Dickeya spp. strains isolated from potato and river water in Finland. Eur J Plant Pathol 126:249–262Google Scholar
  19. 19.
    Lin BR, Shen HF, Pu XM, Tian XS, Zhao WJ, Zhu SF, Dong MM (2010) First report of a soft rot of banana in mainland China caused by a Dickeya sp. (Pectobacterium chrysanthemi). Plant Dis 94:640Google Scholar
  20. 20.
    Martinez LR, Casadevall A (2007) Cryptococcus neoformans biofilm formation depends on surface support and carbon source and reduces fungal cell susceptibility to heat, cold, and UV light. Appl Environ Microbiol 73:4592–4601Google Scholar
  21. 21.
    Murugan K, Selvanayaki K, Al-Sohaibani S (2016) Urinary catheter indwelling clinical pathogen biofilm formation, exopolysaccharide characterization and their growth influencing parameters. Saudi J Biol Sci 23:150–159Google Scholar
  22. 22.
    Navarrete F, de La Fuente L (2014) Response of Xylella fastidiosa to zinc: decreased culturability, increased exopolysaccharide production, and formation of resilient biofilms under flow conditions. Appl Environ Microbiol 80:1097–1107Google Scholar
  23. 23.
    Palacio-Bielsa A, Mosquera MER, Álvarez MAC, Rodríguez IMB, López-Solanilla E, Rodríguez-Palenzuela P (2010) Phenotypic diversity, host range and molecular phylogeny of Dickeya isolates from Spain. Eur J Plant Pathol 127:311–324Google Scholar
  24. 24.
    Prigent-Combaret C, Zghidi-Abouzid O, Effantin G, Lejeune P, Reverchon S, Nasser W (2012) The nucleoid-associated protein Fis directly modulates the synthesis of cellulose, an essential component of pellicle–biofilms in the phytopathogenic bacterium Dickeya dadantii. Mol Microbiol 86:172–186Google Scholar
  25. 25.
    Rinaudi L, Fujishige NA, Hirsch AM, Banchio E, Zorreguieta A, Giordano W (2006) Effects of nutritional and environmental conditions on Sinorhizobium meliloti biofilm formation. Res Microbiol 157:867–875Google Scholar
  26. 26.
    Rodrigues CM, Takita MA, Coletta-Filho HD, Olivato JC, Caserta R, Machado MA, de Souza AA (2008) Copper resistance of biofilm cells of the plant pathogen Xylella fastidiosa. Appl Microbiol Biotechnol 77:1145–1157Google Scholar
  27. 27.
    Shriner AD, Andersen PC (2014) Effect of oxygen on the growth and biofilm formation of Xylella fastidiosa in liquid media. Curr Microbiol 69:866–873Google Scholar
  28. 28.
    Stepanović S, Vuković D, Dakić I, Savić B, Švabić-Vlahović M (2000) A modified microtiter-plate test for quantification of Staphylococcal biofilm formation. J Microbiol Methods 40:175–179Google Scholar
  29. 29.
    Teh AH, Lee SM, Dykes GA (2016) The influence of prior modes of growth, temperature, medium, and substrate surface on biofilm formation by antibiotic-resistant Campylobacter jejuni. Curr Microbiol 73:859–866Google Scholar
  30. 30.
    Tsror L, Erlich O, Lebiush S, Hazanovsky M, Zig U, Slawiak M (2009) Assessment of recent outbreaks of Dickeya sp. (syn. Erwinia chrysanthemi) slow wilt in potato crops in Israel. Eur J Plant Pathol 123:311–320Google Scholar
  31. 31.
    Vinod KK, Lall C, Vimal RR, Vedhagiri K, Sunish IP, Vijayachari P (2016) In vitro antimicrobial susceptibility of pathogenic Leptospira biofilm. Microb Drug Resist 22(7):511–514Google Scholar
  32. 32.
    Wu PH, Huang DD, Chang DCN (2011) Mycorrhizal symbiosis enhances Phalaenopsis orchid’s growth and resistance to Erwinia chrysanthemi. Afr J Biotechnol 10:10095–10100Google Scholar
  33. 33.
    Xu Z, Liang Y, Lin S, Chen D, Li B, Li L, Deng Y (2016) Crystal violet and XTT assays on Staphylococcus aureus biofilm quantification. Curr Microbiol 73(4):1–9Google Scholar
  34. 34.
    Yaganza ES, Tweddell RJ, Arul J (2014) Postharvest application of organic and inorganic salts to control potato (Solanum tuberosum L.) storage soft rot: plant tissue-salt physicochemical interactions. J Agric Food Chem 62:9223–9231Google Scholar
  35. 35.
    Yuan J, Chen Y, Zhou G, Chen H, Gao H (2013) Investigation of roles of divalent cations in Shewanella oneidensis pellicle formation reveals unique impacts of insoluble iron. Biochim Biophys Acta 1830:5248–5257Google Scholar
  36. 36.
    Zaini PA, Fogaça AC, Lupo FG, Nakaya HI, Vêncio RZ, da Silva AM (2008) The iron stimulon of Xylella fastidiosa includes genes for type IV pilus and colicin V-like bacteriocins. J Bacteriol 190:2368–2378Google Scholar
  37. 37.
    Zhang J, Shen H, Pu X, Lin B, Hu J (2014) Identification of Dickeya zeae as a causal agent of banana soft rot in banana in China. Plant Dis 98:436–442Google Scholar
  38. 38.
    Zhang JX, Lin BR, Shen HF, Pu XM (2013) Genome Sequence of the banana pathogen Dickeya zeae strain MS1, which caused bacterial soft rot. Genome Announc 1:e00317–e00313Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Ning Huang
    • 1
  • Xiaoming Pu
    • 2
  • Jingxin Zhang
    • 2
  • Huifang Shen
    • 2
  • Qiyun Yang
    • 2
  • Zhongwen Wang
    • 1
  • Birun Lin
    • 1
    • 2
    Email author
  1. 1.College of AgricultureGuangxi UniversityNanningChina
  2. 2.Guangdong Key Laboratory of High Technology for Plant Protection, Institute of Plant ProtectionGuangdong Academy of Agricultural SciencesGuangzhouChina

Personalised recommendations