Plant and Soil

, Volume 425, Issue 1–2, pp 43–55 | Cite as

Use of a gnotobiotic plant assay for assessing root colonization and mineral phosphate solubilization by Paraburkholderia bryophila Ha185 in association with perennial ryegrass (Lolium perenne L.)

  • Pei-Chun Lisa Hsu
  • Maureen O’Callaghan
  • Leo Condron
  • Mark R. H. Hurst
Methods Paper



The mechanisms by which rhizosphere bacteria increase the availability of mineral P precipitates for plant use are understudied. However, Paraburkholderia bryophila Ha185 is known to solubilize inorganic phosphate in vitro via a novel process. Therefore, this study aimed to demonstrate P solubilization by Ha185 in association with roots of perennial ryegrass (Lolium perenne L.).


We developed a gnotobiotic plant assay to assess P solubilization by Ha185 on ryegrass roots under various nutrient conditions. A green fluorescent protein (GFP)-tagged derivative of Ha185 was used in conjunction with fluorescent microscopy and confocal microscopy to visualize colonization of ryegrass roots.


Ha185 solubilized mineral P (hydroxyapatite) in association with ryegrass roots and increased ryegrass growth by 20% under P-limited conditions. The GFP-tagged Ha185 strain colonized the rhizoplane and penetrated the primary root of ryegrass, possibly through “crack entry” at the point of lateral root emergence, but also by entering the epidermal cells via root hairs.


Ha185 supported ryegrass growth under P-limited conditions, indicating this strain may improve availability of soil P for uptake by ryegrass. Tools developed in this study have broad application in the study of rhizobacteria-plant interactions.


Paraburkholderia Phosphate solubilization Rhizosphere colonization Gnotobiotic plant assay Ryegrass 



Epidermal cells


Green fluorescent protein




Maturation zone


Phosphate-solubilizing bacteria


Rhizodermis cells


Root emergence


Root hairs


Root stele



We thank Manfred Ingerfeld (University of Canterbury, New Zealand) for assistance with confocal microscopy, Aurelie Laugraud (AgResearch, New Zealand) for undertaking 16S rRNA gene analysis, and Pauline Hunt (AgResearch, New Zealand) for assistance with figures. We also thank Tamsin Sheen, PhD, for reviewing and editing a draft of this manuscript.


This work was funded by a grant from the New Zealand Ministry of Business, Innovation and Employment (Grant No. C10X0904).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11104_2018_3633_MOESM1_ESM.docx (262 kb)
Supplemental Figure 1 (DOCX 261 kb)
11104_2018_3633_MOESM2_ESM.avi (19.2 mb)
Supplemental Video S1 (AVI 19656 kb)


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Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Pei-Chun Lisa Hsu
    • 1
    • 2
  • Maureen O’Callaghan
    • 1
    • 2
  • Leo Condron
    • 2
  • Mark R. H. Hurst
    • 1
    • 2
  1. 1.Forage Science, AgResearch LimitedChristchurchNew Zealand
  2. 2.Bio-Protection Research CentreLincoln UniversityLincolnNew Zealand

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