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.)
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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.
KeywordsParaburkholderia Phosphate solubilization Rhizosphere colonization Gnotobiotic plant assay Ryegrass
Green fluorescent protein
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.
- Castanheira N, Dourado AC, Kruz S, Alves P, Delgado-Rodríguez A, Pais I, Semedo J, Scotti-Campos P, Sánchez C, Borges N, Carvalho G, Barreto Crespo MT, Fareleira P (2016) Plant growth-promoting Burkholderia species isolated from annual ryegrass in Portuguese soils. J Appl Microbiol 20:724–739CrossRefGoogle Scholar
- Dower WJ, Miller JF, Ragsdale CW (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucl Acids Res 16:6127–6145Google Scholar
- Holman J, Bugbee B, Chard J (2005) A comparison of coconut coir and sphagnum peat as soil-less media components for plant growth Hydroponics/Soilless Media. Paper 1. https://digitalcommons.usu.edu/cpl_hydroponics/1
- Hsu PC (2014) Determination of genes involved in bacterial phosphate solubilisation. Doctoral dissertation, Lincoln UniversityGoogle Scholar
- Luo S, Xu T, Chen L, Chen J, Rao C, Xiao X, Wan Y, Zeng G, Long F, Liu C, Liu Y (2012) Endophyte-assisted promotion of biomass production and metal-uptake of energy crop sweet sorghum by plant-growth-promoting endophyte Bacillus sp. SLS18. Appl Microbiol Biotechnol 93:1745–1753CrossRefPubMedGoogle Scholar
- Mitter B, Petric A, Shin MW, Chain PS, Hauberg-Lotte L, Reinhold-Hurek B, Nowak J, Sessitsch A (2013) Comparative genome analysis of Burkholderia phytofirmans PsJN reveals a wide spectrum of endophytic lifestyles based on interaction strategies with host plants. Front Plant Sci 4:120CrossRefPubMedPubMedCentralGoogle Scholar
- Nehra V, Choudhary M (2015) A review on plant growth promoting rhizobacteria acting as bioinoculants and their biological approach towards the production of sustainable agriculture. J Appl. Nat Sci 7:540–556Google Scholar
- Oteino N, Lally RD, Kiwanuka S, Lloyd A, Ryan D, Germaine KJ, Dowling DN (2015) Plant growth promotion induced by phosphate solubilizing endophytic Pseudomonas isolates. Front Microbiol 6. https://doi.org/10.3389/fmicb.2015.00745
- Sambrook J, Russell DW (2001) Molecular cloning. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
- Sessitsch A, Coenye T, Sturz AV, Vandamme P, Barka EA, Salles JF, Van Elsas JD, Faure D, Reiter B, Glick BR, Wang-Pruski G, Nowak J (2005) Burkholderia phytofirmans sp. nov., a novel plant-associated bacterium with plant-beneficial properties. Int J Syst Evol Microbiol 55:1187–1192CrossRefPubMedGoogle Scholar
- Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739Google Scholar
- Vandamme P, Opelt K, Knöchel N, Berg C, Schönmann S, De Brandt E, Eberl L, Falsen E, Berg G (2007) Burkholderia bryophila sp. nov. and Burkholderia megapolitana sp. nov., moss-associated species with antifungal and plant-growth-promoting properties. Int J Sys Evol Microbiol 57:2228–2235CrossRefGoogle Scholar
- Versalovic J, Schneider M, De Bruijn FJ, Lupski JR (1994) Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Method. Mol Cell Biol 5:25–40Google Scholar