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Detection of GFP-labeled Paenibacillus polymyxa in autofluorescing pine seedling tissues

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Abstract

Paenibacillus polymyxa P2b-2R is a bacterium that originated from internal lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) seedling stem tissue and fixes nitrogen (N) in association with pine and western red cedar (Thuja plicata Donn.). To evaluate endophytic colonization by this microorganism, we generated P. polymyxa P2b-2Rgfp, a green fluorescent protein (GFP)-labeled derivative of P2b-2R, and grew pine seedlings that were inoculated with the marked strain in a N-limited soil. Tissue disintegration during sample preparation precluded examination of needles for the GFP-labeled endophyte but GFP was detected on roots and in stems of 2- to 14-week-old pine seedlings using confocal laser scanning microscopy. Due to excessive autofluorescence of seedling tissues, labeled bacteria were clearly discernible only in stem tissues of 4- and 6-week-old seedlings. P2b-2Rgfp colonized the root surface extensively and was detected inside the stem cortex, primarily intracellularly. Some labeled bacteria appeared to contain endospores and none were detected in vascular tissues. We conclude that P. polymyxa P2b-2R is capable of endophytic colonization of pine seedlings with specific colonization sites that include the stem cortex but that GFP labeling is of limited value for localization of endophytic bacteria in pine seedling tissues.

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Acknowledgments

Funding for this study was provided by an NSERC Graduate Scholarship and Ministry of Advanced Education of British Columbia (Pacific Leaders Graduate Student Fellowship) to RA and an NSERC Discovery Grant to CPC. We are deeply indebted to Dr. M Itaya for gifting us plasmid pBSGV104 for this study.

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  1. Richa Anand

    Present address: BC Children’s Hospital, 4480 Oak Street, Vancouver, BC, Canada, V6H 3V4

Authors and Affiliations

  1. Department of Forest Sciences, University of British Columbia, 3041-2424 Main Mall, Vancouver, BC, Canada, V6T 1Z4

    Richa Anand & Christopher Peter Chanway

  2. Faculty of Land and Food Systems, University of British Columbia, 248-2357 Main Mall, Vancouver, BC, Canada, V6T 1Z4

    Christopher Peter Chanway

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  1. Richa Anand
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  2. Christopher Peter Chanway
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Correspondence to Christopher Peter Chanway.

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Anand, R., Chanway, C.P. Detection of GFP-labeled Paenibacillus polymyxa in autofluorescing pine seedling tissues. Biol Fertil Soils 49, 111–118 (2013). https://doi.org/10.1007/s00374-012-0727-9

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  • DOI: https://doi.org/10.1007/s00374-012-0727-9

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