Surprising niche for the plant pathogen Pseudomonas syringae

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Abstract

The biology and ecology of plant pathogenic bacteria have been studied almost exclusively in agricultural contexts. In contrast, for numerous human pathogens their biological activity in niches outside of medical contexts is well-known. Whereas there is increasing evidence that traits fostering survival in ‘environmental’ niches can be the basis for virulence factors of human pathogens, niches for plant pathogenic bacteria outside of plants or of agricultural settings have not been elucidated. Most phytopathogenic bacteria are not obligate parasites, some of them can be transported to altitudes of several kilometres, they are scrubbed from the atmosphere by rainfall, and thus they are presumably transported to and might survive in a wide range of habitats. We isolated Pseudomonas syringae from river epilithon (rock-attached biofilms composed of algae, diatoms, rotifers, bacteria and nematodes) at densities up to 6000 cells g−1 in France and the USA, some in pristine settings where waters flowed directly from snow melt and had not passed through agricultural zones. These strains induced hypersensitivity in indicator plants (tobacco) suggesting the presence of functional pathogenicity systems, and many induced disease in 1–7 of the plant species tested and produced a syringomycin-like toxin. Strains also were resistant to some antibiotics used to control plant diseases but not to copper sulphate. Sequencing of the 16S rDNA of epilithon strains and of reference strains of P. syringae revealed that a genetic lineage containing the strains with the broadest host range was distributed across several continents. Is it likely that wide spread dissemination of P. syringae occurs via aerosols and precipitation. This work highlights our limited understanding of non-agricultural niches in the ecology and evolution of plant pathogenic bacteria, of their role in the development of agricultural epidemics both as sources of inoculum and as sources of novel traits that may enhance bacterial pathogenicity and fitness.

Introduction

A recent report by the American Academy of Microbiology (Cangelosi et al., 2004) describes the range of micro-organisms commonly associated with the ‘environment’ at large (aquatic and soil milieu, for example) and with wild or domesticated animals that are also capable of causing significant diseases of humans. That report is part of a growing body of evidence that many human pathogens, including Vibrio cholera, Burkholderia cepacia, Legionnella pneumophila, Mycobacterium avium and Pseudomonas aeruginosa, have acquired some of their virulence factors as well as resistance to antibiotics in their environmental (non-human host) niches and that strategies for growth and survival in these niches can accord advantages for infecting human hosts. V. cholera is particularly illustrative of the notion that some virulence mechanisms may result from adaptations made for effective colonisation and survival in environmental niches. The toxin-coregulated pilus of V. cholera, an essential virulence factor, has been demonstrated to be also involved in biofilm differentiation on chitinaceous surfaces and in overall ecological fitness (Reguera and Kolter, 2005), revealing an important biological link between this bacterium's capacity for association with copepods in aquatic environments and the acquisition of traits essential for virulence to humans. Likewise, it has been proposed that virulence determinants in P. aeruginosa can arise in environmental niches through selective pressures for effective efflux pumps and mechanisms for translocating exoenzymes that are unrelated to the use of antibiotics or to the human host–pathogen interaction (Alonso et al., 1999).

In contrast to medically important micro-organisms, the life cycles and biology of plant pathogens have been almost exclusively defined in agricultural contexts sensu stricto and in terms of the availability and sensitivity of host plants. Sources of inoculum for epidemics are generally sought in plant tissues and debris, on agricultural equipment and structures, in insects as possible vectors, and in irrigation waters. The notions of biodiversity and evolution of plant pathogens are based almost entirely on collections from cultivated host plants (McDonald and Linde, 2002, Poussier et al., 2000, Rademaker et al., 2000, Sarkar and Guttman, 2004) and comparatively few studies of diseases of wild plants (Antonovics et al., 2002, Gilbert, 2002). These investigations have rendered considerable fruit in terms of our understanding of disease spread and for developing strategies to select for disease-resistant plant varieties and for other prophylactic measures for disease control. Yet, it is likely that we are missing a potentially significant part of the picture of the life of plant pathogens. Studies of population genetics of pathogens can be considerably biased if they are based exclusively on strains from disease epidemics (Spratt and Maiden, 1999). Furthermore, agricultural systems are open and most plant pathogens are not obligate parasites. Many plant pathogens can be aerially disseminated considerable distances. The notion that plant pathogens have ‘other lives’ and the impact that adaptation to niches other than those provided by plants (cultivated or wild) could have on virulence, pathogen fitness and emergence of new pathotypes has not been explored, primarily because these niches have not been identified or described.

Species known to be plant pathogens have been detected in a wide range of environmental settings. The aerially dispersed broad host range fungus Botrytis cinerea, for example, has been detected on stone monuments and in rocks (Burford et al., 2003, Gorbushina et al., 2002) in stored hydrocarbon fuels (Gaylarde et al., 1999) and on the hair of small mammals and of school children (Ali-Shtayeh et al., 2000, Shchipanov et al., 2003). Via direct characterization of DNA from environmental samples, Ralstonia solanacearum has been reported from lake sediments in copper-mining regions (Konstantinidis et al., 2003). Pectolytic enzyme-producing strains of Erwinia chrysanthemi have been collected from water in the alpine sources of two major river systems in Australia and are thought to be part of the indigenous micro-organisms on weeds and in sediments in these alpine streams (Cother and Gilbert, 1990). However, the phytopathogenicity of none of the organisms listed above has been investigated for environmental samples.

Pseudomonas syringae, a plant pathogen with world-wide economic importance that causes necroses on leaves, stems, fruits and other aerial plant parts, is especially likely to exist in diverse niches. This bacterium can establish large populations on aerial surfaces of a wide range of plant species without necessarily causing disease (Hirano and Upper, 1990). From this epiphytic phase it can be disseminated via aerosols. Upward movement of P. syringae is an important component of its dissemination, with net upward flux from plant canopies being on the order of 105 cells m−2 h−1 (Lindemann et al., 1982). P. syringae is one of the few plant pathogens known to be disseminated up into clouds (Jayaweera and Flanagan, 1982, Sands et al., 1982). It is also scrubbed from the air during rain (Constantinidou et al., 1990). Many strains of this bacterium are ice nucleation-active (Lindow, 1983), are known to survive freezing as well as induce freezing, and it has been suggested that they might even have a role in inciting rainfall via their ice nucleation activity (Morris et al., 2004). P. syringae has been shown to survive in lake water (Riffaud and Morris, 2002) and therefore seems likely to survive in other types of fresh water. As a plant pathogen it is adapted to exploiting photosynthates of host plants. Some strains also produce syringomycin and related toxins to which a wide range of prokaryotes and eukaryotic cells are sensitive (Bender et al., 1999). Taken together, these diverse properties suggest that it is likely that P. syringae is disseminated to and survives and multiplies in a wide range of habitats and niches.

Processes associated with global water cycling might be a particularly important vehicle for P. syringae dissemination. In particular, this bacterium could fall into streams and rivers with rain or snowmelt as well as with run-off that carries debris from infected plants. Flow rates of small streams (ca. 1 m3 s−1) could carry nearly 105 P. syringae each day across rocks at a given site in a stream if the water contained as little as one cell of this bacterium per m3, for example. River epilithon (rock-attached biofilms that are commonly composed of algae, diatoms, rotifers, bacteria and nematodes) could trap P. syringae cells from flowing water. The combination of continuous moisture and availability of photosynthates from algae might offer the basis for a suitable habitat for growth and persistence. Based on this reasoning, we attempted to isolate P. syringae from epilithon at diverse sites. Here we report the biological properties – including host range, resistance to biocides used in agriculture, ice nucleation activity and toxin production – and the genetic diversity of the P. syringae and related pathogens that were isolated.

Section snippets

Origin of strains

Biofilms were scraped from rocks using sterile spatulas and transported to the lab in sterile containers. Sampling sites (and dates) were in the Vaucluse district of France in (1) the Auzon River at Notre Dame de Pareloup (January 2005) and (2) the Sorgues River at Fontaine de Vaucluse (January 2005); (3) Gallatin County, Montana, USA in the east fork of Hyalite Creek at Palisade Falls (September 2005); (4) Park County, Montana in Pine Creek at Pine Creek Falls (September 2005) and (5) Mill

Results and discussion

Bacteria were isolated from epilithon in five streams and rivers in Montana, Utah and southern France. The sampling sites in Montana were in pristine forests that could be accessed only on foot and were upstream of agricultural fields. Aliquots of all samples were observed microscopically revealing that they contained typical components of river epilithon including diatoms, rotifers, nematodes, copepods, algae and bacteria. At some of the sampling sites, moss and other plants were also attached

Acknowledgements

Funding for K.X. was provided by the US National Science Foundation Microbial Observatories project 9977907 to L.K. We thank Dr. Anne Camper and Dr. Andreas Nocker, Center for Biofilm Engineering, Montana State University, for fruitful discussions about pathogen ecology and for technical advice. We thank Catherine Glaux of INRA-Avignon for technical help.

References (45)

  • S.F. Altschul et al.

    Basic local alignment search tool

    J. Mol. Biol.

    (1990)
  • E.P. Burford et al.

    Geomycology: fungi in mineral substrata

    Mycologist

    (2003)
  • T.H. Jukes et al.

    Evolution of protein molecules

  • G. Vali

    Ice nucleation—a review

  • M.S. Ali-Shtayeh et al.

    Hair and scalp mycobiota in school children in Nablus area

    Mycopathologia

    (2000)
  • A. Alonso et al.

    Environmental and clinical isolates of Pseudomonas aeruginosa show pathogenic and biodegrative properties irrespective of their origin

    Environ. Microbiol.

    (1999)
  • J. Antonovics et al.

    The ecology and genetics of a host shift: Microbotryum as a model system

    Am. Nat.

    (2002)
  • C.L. Bender et al.

    Pseudomonas syringae phytotoxins: Mode of action, regulation and biosynthesis by peptide and polyketide synthetases

    Microbiol. Molec. Biol. Rev.

    (1999)
  • Cangelosi, G.A., Freitag, N.E., Buckley, M.R., 2004. From outside to inside: environmental microorganisms as human...
  • P. Caruso et al.

    Seasonal variation of Ralstonia solanacearum biovar 2 populations in a Spanish river: recovery of stressed cells at low temperatures

    Appl. Environ. Microbiol.

    (2005)
  • H.A. Constantinidou et al.

    Atmospheric dispersal of ice nucleation-active bacteria: the role of rain

    Phytopathology

    (1990)
  • E.J. Cother et al.

    Presence of Erwinia chrysanthemi in two major river systems and their alpine sources in Australia

    J. Appl. Bacteriol.

    (1990)
  • U. Edwards et al.

    Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA

    Nucleic Acids Res.

    (1989)
  • J. Felsenstein

    PHYLIP Version 3.6. Software Distributed by the Author

    (2005)
  • C.C. Gaylarde et al.

    Microbial contamination of stored hydrocarbon fuels and its control

    Revista de Microbiologia

    (1999)
  • G.S. Gilbert

    Evolutionary ecology of plant diseases in natural ecosystems

    Annu. Rev. Phytopathol.

    (2002)
  • A.A. Gorbushina et al.

    Microbial communities on the monuments of Moscow and St. Petersburg: biodiversity and trophic relations

    Mikrobiologiya

    (2002)
  • D.C. Gross

    Regulation of syringomycin synthesis in Pseudomonas syringae pv. syringae and defined conditions for its production

    J. Appl. Bacteriol.

    (1985)
  • D.C. Gross et al.

    Population dynamics and pathogenesis of Pseudomonas syringae in maize and cowpea in relation to the in vitro production of syringomycin

    Phytopathology

    (1977)
  • S.Y. He

    Elicitation of plant hypersensitive response by bacteria

    Plant Physiol.

    (1996)
  • S.S. Hirano et al.

    Population biology and epidemiology of Pseudomonas syringae

    Annu. Rev. Phytopathol.

    (1990)
  • K. Jayaweera et al.

    Investigation of biogenic ice nuclei in the Arctic atmosphere

    Geophys. Res. Lett.

    (1982)
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