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Development of a new technique to detect living cells of Xanthomonas campestris pv. campestris in crucifers seeds: the seed-qPCR

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Abstract

Xanthomonas campestris pv. campestris is a seed-borne bacterium that causes black rot on Brassicaceae. Ensuring seed lot sanitary quality is the most efficient control strategy against bacterial diseases. Currently, the procedures adopted in the control of seed lots are mainly based on microbiological techniques combined with PCR or plant inoculation which is time and money consuming. The aim of this study was to propose a reliable and rapid detection technique of living X. c. pv. campestris in cabbage seeds. We have shown that not all cells of X. campestris is able to grown on rich medium after washing and soaking seeds as no colony of X. campestris was detected on inoculated seeds, whereas plantlets develop symptoms 7–14 days after germination. The PCR technique used alone does not address the viability of bacteria in samples. We set up a technique named seed-qPCR for the detection of living X. c. pv. campestris bacterial cells in seed lots. This technique is based on an enrichment of bacterial population associated with infected seeds by seed germination coupled with real-time Taq-man PCR after extraction of the target DNA. It is an inexpensive technique that allow the detection of down to 1 contaminated seed among 10 000 healthy seeds. The seed-qPCR method combines an efficient extraction based on bacterial multiplication on seedlings with a sensitive technique qPCR for the detection of bacteria in seed lots.

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Acknowledgments

We thank Valérie Grimault, Geves-SNES, France, for her kind provision of seeds.

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Authors and Affiliations

  1. CNCC, Centre National de Contrôle et de Certification des semences et plants, BP 119 Hassen Badi, 16200, El Harrach, Alger, Algeria

    Samia Laala

  2. ENSA, Ecole Nationale Supérieure d’Agriculture, 1 Avenue Pasteur, Hassan Badi - El Harrach - Alger, 16000, El Harrach, Alger, Algeria

    Zouaoui Bouznad

  3. Anses, Laboratoire de la santé des végétaux, 7 rue Jean Dixméras, 49044, Angers, France

    Charles Manceau

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  1. Samia Laala
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  2. Zouaoui Bouznad
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  3. Charles Manceau
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Correspondence to Charles Manceau.

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Laala, S., Bouznad, Z. & Manceau, C. Development of a new technique to detect living cells of Xanthomonas campestris pv. campestris in crucifers seeds: the seed-qPCR. Eur J Plant Pathol 141, 637–646 (2015). https://doi.org/10.1007/s10658-014-0532-4

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  • DOI: https://doi.org/10.1007/s10658-014-0532-4

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