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Niches and routes of transmission of Xanthomonas citri pv. fuscans to bean seeds

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Abstract

Aims

Seeds are vectors of a diversified microbiota including plant pathogens. To better understand transmission of common bacterial blight (CBB) agents to bean seeds, we analyzed the role of non-pathogenic xanthomonads on seed transmission efficiency and investigated the location of Xanthomonas citri pv. fuscans (Xcf) into seeds and plantlets.

Methods

Competition between CBB and NP strains was initially assessed in vitro and then extended in planta to monitor the impact of co-inoculation on Xcf seed transmission. Moreover, location of Xcf strains in seeds and seedlings was visualized using a combination of gfp-tagged strain and DOPE-FISH/CSLM.

Results

Whereas CBB agent growth was inhibited in vitro by some seed-borne non-pathogenic xanthomonads strains, these strains did not transmit efficiently to seed through floral pathway and did not affect Xcf seed transmission. Xcf cells were observed entering seed through vascular elements and parenchyma of funiculus, but also micropyle and testa. Xcf cells were observed, moreover, among other bacteria on radicle surfaces, especially tip, in cotyledons, and plumules.

Conclusions

CBB agents are more efficient than non-pathogenic xanthomonads in using the floral route to colonize seeds. CBB agents are located within different niches in the seed tissues up to the embryonic axis.

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Abbreviations

Xcf :

Xanthomonas citri pv. fuscans

GFP:

Green fluorescent protein

NP:

Non-pathogenic

CBB:

Common bacterial blight

DOPE-FISH:

Double labeling of oligonucleotide probes for fluorescence in situ hybridization

CSLM:

Confocal laser-scanning microscopy

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Acknowledgements

We thank Jean-François Guimbaud for his participation in the experiments reported here. His salary and part of this work were supported by the European Commission (TESTA, FP7-KBBE-2012-6, 311875). We thank Marjorie Juchaux and Mayeul Milien from the IMAC facility of SFR 4207 Quasav for microscopy support, David Logan (IRHS) for providing plasmids carrying the GFP and mCherry cassettes, CIRM-CFBP (Beaucouzé, INRA, France; http://www6.inra.fr/cirm_eng/CFBP-Plant-Associated-Bacteria) for strain preservation and supply; Céline Rousseau and Daniel Sochard from the Phenotic platform facility of SFR 4207 Quasav for support with fluorescence-chlorophyll imaging/data analysis and plant cultivation, respectively.

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Authors and Affiliations

  1. IRHS, INRA, AGROCAMPUS-Ouest, Université d’Angers, SFR4207 QUASAV, IRHS 42, CS60057, rue Georges Morel, 49071, Beaucouzé cedex, France

    Armelle Darrasse, Matthieu Barret, Sophie Cesbron & Marie-Agnès Jacques

  2. AIT Austrian Institute of Technology GmbH, Center for Health and Bioresources, Bioresources Unit, Konrad Lorenz Straße 24, A-3430, Tulln, Austria

    Stéphane Compant

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  1. Armelle Darrasse
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  2. Matthieu Barret
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  3. Sophie Cesbron
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  5. Marie-Agnès Jacques
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Correspondence to Marie-Agnès Jacques.

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Matthieu Barret and Stéphane Compant are Guest Editors of the special issue. This does not, however, interfere with the reviewing process.

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Responsible Editor: Birgit Mitter.

Electronic supplementary material

Table S1

(DOCX 19 kb)

Fig. S1

Population sizes of pathogenic and non-pathogenic bacterial strains following vacuum-infiltration of seeds (a), in 4d–old seedlings (b), and in cataphylls of 7 d-old plantlets (c). Bacterial strains were inoculated alone or in mixture (1:1) by vacuum-infiltration and quantified 2 h following seed inoculation in 4d–old seedlings grown in germination boxes from inoculated seeds, and in cataphylls of 7 d-old plantlets grown in soil. Code of the CBB strain/code of non-pathogenic strain. Means and SEMs were calculated for 10 samples per treatment. Mean population densities followed by different letters are significantly (P < 0.05) different based on the Mann-Whitney test. (PPTX 2945 kb)

Fig. S2

In planta stability of plasmids containing the marker genes (gfp or mCherry) in four strains representing the genetic diversity of common bacterial blight agents. Strain 7767-R representing Xcf, strain 6988-R representing Xcf NF2, strain 6996-R representing Xcf NF3, and strain 6937-R representing Xpp were transformed with pBBR1MCS-2 (A), pBBR1MCS-2::gfp6 (B), pBBR1MCS-5::mCherry (C), and pBBR1MCS-5 (D) Population sizes of strains were quantified in selective (black) and non-selective (grey) media nine days post-inoculation of bean leaves. Means and SEMs were calculated for three leaflets per treatment. Differences in population sizes were not significant (P < 0.05) between each transformant and the wild type, and for each transformant, between the population size determined from selective and non-selective media, on the basis of the Mann-Whitney test (PPTX 161 kb)

Fig. S3

CSLM of a developing seed (17d–old) at the micropyle level. In these frontal sections Xcf::gfp bacteria were located on the surface of the seed (3D reconstruction of a series of confocal images (a), within the seed tissues in the parenchyma (b), in the micropyle cavity (c), below the hilum within the tracheid bar and in the surrounding parenchyma (d), in the parenchyma below the lens (e), in the parenchyma deeper in the seed (f), and in the light-tight embryo tissues, between disjointed cells (g and h), as highlighted by the zoom area panel h. CSLM images were generated by merging channels 488 nm and transmitted light (a to g) or under hyperspectral detector mode (excitation at 488 and 405 nm and signal reception with all channels) (h). White boxes correspond to a three times-magnification of the selected area; white arrows highlight GFP tagged-cells; tissues or structures are indicated by letters: area under micropyle (um), hilar scar (hs), tracheid bar (tb) (PPTX 20229 kb)

Fig. S4

CSLM images of Xcf::gfp in a dried symptomatic seed (40 d-old). The epidermal layer of macrosclereids was interrupted at several places by GFP-tagged bacterial cells invading testa parenchyma and cotyledons. Images of transverse section of seeds were generated by merging channels 488 nm and transmitted light (a) and under hyperspectral detector mode (excitation at 488 and 405 nm and signal reception with all channels, b). White arrows highlight GFP tagged-cells; tissues or structures are indicated by letters: testa parenchyma (tp), palisade of macrosclereids (pm), embryo (em) (PPTX 4764 kb)

Fig. S5

CSLM images of Xcf::gfp in transverse section of a symptomatic contaminated seed (40 d-old) after imbibition during 24 h. GFP-tagged cells were located on the surface of cotyledons and embryo (a, b), in bacterial aggregates at the surface of the hypocotyle (c, d), and in-between embryo folds but external from embryo tissues (e, f). Confocal plans of the the plumule tissues showed external location of GFP-tagged cells (g, h). Images were generated by merging channels 488 nm and transmitted light (a, c, e, g, h) or under hyperspectral detector mode (excitation at 488 and 405 nm and signal reception with all channels). White boxes correspond to a three times-magnification of the selected area; white arrows highlight GFP tagged-cells (PPTX 21458 kb)

Fig. S6

CSLM images of Xcf::gfp in seedlings. Seeds were inoculated by inoculum deposit over the hilum area including micropyle and lens. GFP-tagged cells were present at the surface of the radicle of 3 d-old seedlings (a, b), in aggregates at the point of emergence of radicle (c, d), and at the bases of trichomes on the external surface of plumule of a 6 d-old seedling. Images of transverse sections were generated by merging channels 488 nm and transmitted light (a, c, e) or under hyperspectral detector mode (excitation at 488 and 405 nm and signal reception with all channels, b, d, f). White boxes correspond to a three times-magnification of the selected area; white arrows highlight GFP tagged-cells (PPTX 15129 kb)

Fig. S7

CSLM microphotographs of seeds infested or not with Xcf 7767-R visualized with NONEUB probes with ATT0488 and Cy5 showing negative signals (PPTX 453 kb)

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Darrasse, A., Barret, M., Cesbron, S. et al. Niches and routes of transmission of Xanthomonas citri pv. fuscans to bean seeds. Plant Soil 422, 115–128 (2018). https://doi.org/10.1007/s11104-017-3329-3

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