Wheat seed embryo excision enables the creation of axenic seedlings and Koch's postulates testing of putative bacterial endophytes.

Rebekah J Robinson,Ian M Clark,Robert W Jackson,Penny R Hirsch,Tim H Mauchline,Bart A Fraaije

doi:10.1038/srep25581

Rebekah J Robinson, Ian M Clark + Show 4 more

Open Access

https://doi.org/10.1038/srep25581

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Journal: Scientific Reports	Publication Date: May 1, 2016
Citations: 46	License type: CC BY 4.0

Affiliation: Rothamsted Research, University of Reading

Abstract

Early establishment of endophytes can play a role in pathogen suppression and improve seedling development. One route for establishment of endophytes in seedlings is transmission of bacteria from the parent plant to the seedling via the seed. In wheat seeds, it is not clear whether this transmission route exists, and the identities and location of bacteria within wheat seeds are unknown. We identified bacteria in the wheat (Triticum aestivum) cv. Hereward seed environment using embryo excision to determine the location of the bacterial load. Axenic wheat seedlings obtained with this method were subsequently used to screen a putative endophyte bacterial isolate library for endophytic competency. This absence of bacteria recovered from seeds indicated low bacterial abundance and/or the presence of inhibitors. Diversity of readily culturable bacteria in seeds was low with 8 genera identified, dominated by Erwinia and Paenibacillus. We propose that anatomical restrictions in wheat limit embryo associated vertical transmission, and that bacterial load is carried in the seed coat, crease tissue and endosperm. This finding facilitates the creation of axenic wheat plants to test competency of putative endophytes and also provides a platform for endophyte competition, plant growth, and gene expression studies without an indigenous bacterial background.

Highlights

Cultured bacterial population densities up to 3.5 × 105 CFU g−1 fresh tissue were found in rice seeds and 9 genera were identified by culture dependent analysis and 17 genera through culture independent analysis[29]
The most abundant bacterial strain recovered from wheat seeds was identified as a representative of the genus Erwinia
Bacterial colonies were not recovered from surface sterilised seed macerate indicating absence or very low abundance of culturable bacteria in wheat seeds or the presence of bacterial growth inhibitors in the seed

Summary

Introduction

Ruiza et al.[30] found endophyte densities of up to 5 × 106 CFU g−1 fresh tissue in rice seeds and isolated a number of genera including Pantoea, Curtobacterium, Paenibacillus, Rhizobium and Microbacterium[30]. No detectable bacterial 16S rRNA gene product was amplified from the DNA extractions of culture-sterile samples of roots or shoots of embryo-grown seedlings (Fig. 1). Bacterial colonies were not recovered from surface sterilised seed macerate indicating absence or very low abundance of culturable bacteria in wheat seeds or the presence of bacterial growth inhibitors in the seed.

Results

Conclusion