Microbial Ecology

, Volume 47, Issue 1, pp 87–95 | Cite as

Comparison of the Phenotypes and Genotypes of Biofilm and Solitary Epiphytic Bacterial Populations on Broad-Leaved Endive

  • T. Boureau
  • M. -A. Jacques
  • R. Berruyer
  • Y. Dessaux
  • H. Dominguez
  • C. E. MorrisEmail author


The discovery that biofilms are ubiquitous among the epiphytic microflora of leaves has prompted research about the impact of biofilms on the ecology of epiphytic microorganisms and on the efficiency of strategies to manage these populations for disease control and to ensure food safety. Biofilms are likely to influence the microenvironment and phenotype of the microorganisms they harbor. However, it is also important to determine whether there are differences in the types of bacteria within biofilms compared to those outside of biofilms so as to better target microorganisms via disease control strategies. Broad-leaved endive (Cichorium endivia var. latifolia) harbors biofilms containing fluorescent pseudomonads. These bacteria can cause considerable post-harvest losses when this plant is used for manufacturing minimally processed salads. To determine whether the population structure of the fluorescent pseudomonads in biofilms is different from that outside of biofilms on the same leaves, bacteria were isolated quantitatively from the biofilm and solitary components of the epiphytic population on leaves of field-grown broad-leaved endive. Population structure was determined in terms of taxonomic identities of the bacteria isolated, in terms of genotypic profiles, and in terms of phenotypic traits related to surface colonization and biofilm formation. The results illustrate that there are no systematic differences in the composition and structure of biofilm and solitary populations of fluorescent pseudomonads, in terms of either genotypic profiles or phenotypic profiles of the strains. However, Gram-positive bacteria tended to occur more frequently within biofilms than outside of biofilms. We suggest that leaf colonization by fluorescent pseudomonads involves a flux of cells between biofilm and solitary states. This would allow bacteria to exploit the advantages of these two types of existence; biofilms would favor resistance to stressful conditions, whereas solitary cells could foster spread of bacteria to newly colonizable sites on leaves as environmental conditions fluctuate.


Biosurfactants Fluorescent Pseudomonad Genotypic Profile Epiphytic Bacterium Solitary Cell 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We thank C. Glaux for technical help and the two anonymous reviewers for very pertinent remarks and criticisms of the manuscript.


  1. 1.
    Bayot, RG, Ries, SM 1986Role of motility in apple blossom infection by Erwinia amylovora and studies of fire blight control with attractant and repellent compounds.Phytopathology76441445Google Scholar
  2. 2.
    Bertheau, Y, Madgidi-Hervan, E, Koutoujansky, A, Nguyen-the, C, Andro, T, Coleno, A 1984Detection of depolymerase isoenzymes after electrophoresis or electrofocusing, or in titration curves.Anal Biochem139383389PubMedGoogle Scholar
  3. 3.
    Burrage, SW 1972Dew on wheat.Agric Meteorol10312CrossRefGoogle Scholar
  4. 4.
    Carmichael, I, Harper, IS, Coventry, MJ, Taylor, PWJ, Hickey, MW 1999Bacterial colonization and biofilm development on minimally processed vegetables.J Appl Microbiol Symp Suppl8545S51SGoogle Scholar
  5. 5.
    Characklis, WG, Marshall, KC 1990Biofilms.John Wiley & SonsNew YorkGoogle Scholar
  6. 6.
    Costerton, JW, Cheng, KJ, Geesey, GG, Ladd, TI, Nickel, JC, Dasgupta, M, Marrie, TJ 1987Bacterial biofilms in nature and disease.Ann Rev Microbiol41435464CrossRefGoogle Scholar
  7. 7.
    Costerton, JW, Lewandowski, Z, Caldwell, DE, Korber, DR, Lappinscott, HM 1995Microbial biofilms.Ann Rev Microbiol49711745Google Scholar
  8. 8.
    DeLong, EF, Franks, DG, Alldredge, AL 1993Phylogenetic diversity of aggregate-attached vs. free-living marine bacterial assemblages.Limnol Oceanogr38924934Google Scholar
  9. 9.
    Denny, TP 1999Autoregulator-dependent control of extracellular polysaccharide production in phytopathogenic bacteria.Eur J Plant Pathol105417430CrossRefGoogle Scholar
  10. 10.
    Fett, WF 2000Naturally occurring biofilms on alfalfa and other types of sprouts.J Food Protect63625632Google Scholar
  11. 11.
    Gerhardt, PMurray, RGECostilow, RNNester, EWWood, WAKrieg, NRPhillips, GB eds. 1981Manual of Methods for General Bacteriology.WashingtonDCGoogle Scholar
  12. 12.
    Giesler, LJ, Yuen, GY, Horst, GL 2000Canopy microenvironments and applied bacteria population dynamics in shaded tall fescue.Crop Sci4013251332Google Scholar
  13. 13.
    Hildebrand, DC 1971Pectate and pectin gels for differentiation of Pseudomonas sp. and other bacterial plant pathogens.Phytopathology6114301436Google Scholar
  14. 14.
    Holt, JGKrieg, NRSneath, PHAStaley, JTWilliams, ST eds. 1994Bergey’s Manual of Determinative Bacteriology.Williams and WilkinsBaltimoreGoogle Scholar
  15. 15.
    Jacques, M-A, Kinkel, LL, Morris, CE 1995Population sizes, immigration, and growth of epiphytic bacteria on leaves of different ages and positions of field-grown endive (Cichorium endivia var. latifolia).Appl Environ Microbiol61899906Google Scholar
  16. 16.
    Jacques, M-A, Morris, CE 1995Bacterial population dynamics and decay on leaves of different ages of ready-to-use broad-leaved endive.Int J Food Sci Technol30221236Google Scholar
  17. 17.
    Karthikeyan, S, Wolfaardt, GM, Korber, DR, Caldwell, DE 1999Functional and structural responses of a degradative microbial community to substrates with varying degrees of complexity in chemical structure.Microb Ecol38215224CrossRefPubMedGoogle Scholar
  18. 18.
    King, EO, Ward, MK, Raney, DE 1954Two simple media for the demonstration of pyocyanin and fluorescin.J Lab Clin Med44301307PubMedGoogle Scholar
  19. 19.
    Klement, ZRudolph, KSands, DC eds. 1990Methods in Phytobacteriology.Akademiai KiadoBudapestGoogle Scholar
  20. 20.
    Kolattukudy, PE 1996Biosynthetic pathways of cutin and waxes, and their sensitivity to environmental stresses.Kerstiens, G eds. Plant Cuticles.BIOS Scientific PublishersOxford83108Google Scholar
  21. 21.
    Korber, DR, Lawrence, JR, Sutton, B, Caldwell, DE 1989Effect of laminar flow velocity on the kinetics of surface recolonization by Mot+ and Mot− Pseudomonas fluorescens. Microb Ecol18119Google Scholar
  22. 22.
    Lawrence, JR, Korber, DR, Wolfaardt, GM, Caldwell, DE 1995Behavioral strategies of surface-colonizing bacteria.Adv Microb Ecol14175Google Scholar
  23. 23.
    Liao, CH 1991Cloning of pectate lyaze gene pel from Pseudomonas fluorescens and detection of sequences homologous to pel in Pseudomonas viridiflava and Pseudomonas putida. J Bacteriol17343864393PubMedGoogle Scholar
  24. 24.
    Louws, FJ, Fulbright, DW, Stephens, CT, de Bruijn, FJ 1994Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR.Appl Environ Microbiol6022862295PubMedGoogle Scholar
  25. 25.
    Marques, AS, dos, A, Corbière, R, Gardan, L, Tourte, C, Manceau, C, Taylor, JD, Samson, S 2000Multiphasic approach for the identification of the different classification levels of Pseudomonas savastanoi pv. phaseolicola. Eur J Plant Pathol106715734Google Scholar
  26. 26.
    McLean, RJC, Whiteley, M, Stickler, DJ, Fuqua, WC 1997Evidence of autoinducer activity in naturally occurring biofilms.FEMS Microbiol Lett154259263CrossRefPubMedGoogle Scholar
  27. 27.
    Morris, CE 2001Phyllosphere Encyclopedia for Life Sciences, Publishing GroupLondonGoogle Scholar
  28. 28.
    Morris, CE, Barnes, MB, McLean, RJC 2002Biofilms on leaf surfaces: implications for the biology, ecology and management of populations of epiphytic bacteria.Lindow, SEHecht-Poinar, EIElliot, V eds. Phyllosphere Microbiology.APS PressMinneapolis138154Google Scholar
  29. 29.
    Morris, CE, Kinkel, LL 2002Fifty years of phyllosphere microbiology: Significant contributions to research in related fields.Lindow, SEHecht-Poinar, EIElliot, V eds. Phyllosphere Microbiology.APS PressMinneapolis353363Google Scholar
  30. 30.
    Morris, CE, Monier, J-M, Jacques, M-A 1997Methods for observing microbial biofilms directly on leaf surfaces and recovering them for isolation of culturable microorganism.Appl Environ Microbiol6315701576Google Scholar
  31. 31.
    Morris, CE, Monier, J-M, Jacques, M-A 1998A technique to quantify the population size and composition of the biofilm component in communities of bacteria in the phyllosphere.Appl Environ Microbiol6447894795PubMedGoogle Scholar
  32. 32.
    Nguyen-the, C, Carlin, F 1994The microbiology of minimally processed fresh fruits and vegetables.Crit Rev Food Sci Nutr34371401PubMedGoogle Scholar
  33. 33.
    Prunier, JP, Kaiser, P 1961Etude de l’activité pectinolytique chez des bactéries phytopathogènes et saprophytes des plantes.—I. Recherche des enzymes pectinolytiques.Ann Epiphytes15205219Google Scholar
  34. 34.
    Roine, E, Nunn, DN, Paulin, L, Romantschuk, M 1996Characterization of genes required for pilus expression in Pseudomonas syringae pathovar phaseolicola. J Bacteriol178410417PubMedGoogle Scholar
  35. 35.
    Ross, N, Villemur, R, Marcandella, E, Deschenes, L 2001Assessment of changes in biodiversity when a community of ultramicrobacteria isolated from groundwater is stimulated to form a biofilm.Microb Ecol425668PubMedGoogle Scholar
  36. 36.
    Ryu, E 1940A simple method of differentiating between Gram-positive and Gram-negative organisms without staining.Kitasato Arch Exp Med175863Google Scholar
  37. 37.
    Shaw, PD, Ping, G, Daly, SL, Cha, C, Cronan, JE, Rinehart, KL, Farrand, SK 1997Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography.Proc Natl Acad Sci USA9460366041CrossRefPubMedGoogle Scholar
  38. 38.
    Stanier, RY, Palleroni, NJ, Doudoroff, M 1966The aerobic pseudomonads: a taxonomic study.J Gen Microbiol43159271PubMedGoogle Scholar
  39. 39.
    Tresse, O, Lorrain, MJ, Rho, D 2002Population dynamics of free-floating and attached bacteria in a styrene-degrading biotrickling filter analyzed by denaturing gradient gel electrophoresis.Appl Microbiol Biotechnol59585590CrossRefPubMedGoogle Scholar
  40. 40.
    Versolavic, J, Schneider, M, de Bruijn, FJ, Lupski, JR 1994Genomic fingerprinting of bacteria using repetitive sequence based (rep-PCR).Methods Cell Mol Biol52540Google Scholar
  41. 41.
    Whiteley, M, Ott, JR, Weaver, EA, McLean, RJC 2001Effects of community composition and growth rate on aquifer biofilm bacteria and their susceptibility to betadine disinfection.Environ Microbiol34352CrossRefPubMedGoogle Scholar
  42. 42.
    Zar, JH 1984Biostatistical Analysis.Prentice-HallEnglewood Cliffs, NJGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • T. Boureau
    • 1
  • M. -A. Jacques
    • 2
  • R. Berruyer
    • 3
  • Y. Dessaux
    • 3
  • H. Dominguez
    • 4
  • C. E. Morris
    • 4
    Email author
  1. 1.Division of General MicrobiologyUniversity of Helsinki, Biocenter, P.O. Box 56, HelsinkiFinland
  2. 2.Station de Pathologie VégétaleINRA, 42 rue Georges Morel, B.P. 57, 49071 BeaucouzéFrance
  3. 3.Institut des Sciences du VégétalCNRS, Bat. 23, avenue de la Terrasse, Gif-sur-YvetteFrance
  4. 4.Station de Pathologie VégétaleINRA, BP 94, 84140 MontfavetFrance

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