Development and validation of a real-time quantitative PCR assay to detect Xanthomonas axonopodis pv. allii from onion seed.
Bacterial blight of onion is an emerging disease threatening world onion production. The causal agent Xanthomonas axonopodis pv. allii is seed transmitted and a reliable and sensitive tool is needed to monitor seed exchanges. A triplex quantitative real-time PCR assay was developed targeting two X. axonopodis pv. allii-specific markers and an internal control chosen in 5.8S rRNA gene from Alliaceae. Amplification of at least one marker indicates the presence of the bacterium in seed extracts. This real-time PCR assay detected all the 79 X. axonopodis pv. allii strains tested and excluded 85.2% of the 135 non-target strains and particularly all 39 saprophytic and pathogenic bacteria associated with onion. Cross-reactions were mainly obtained for strains assigned to nine phylogenetically related X. axonopodis pathovars. The cycle cut-off was estimated statistically at 36.3 considering a risk of false positive of 1%. The limit of detection obtained in at least 95% of the time (LOD 95%) was 5×10(3) CFU/g (colony forming unit/g). The sensitivity threshold was found to be 1 infected seed in 32,790 seeds. This real-time PCR assay should be useful for preventing the long-distance spread of X. axonopodis pv. allii via contaminated seed lots and determining the epidemiology of the bacterium.
CIRAD, UMR Peuplements Végétaux et Bioagresseurs en Milieu Tropical, CIRAD-Université de la Réunion, Pôle de Protection des Plantes (3P), 7, chemin de l'IRAT, 97410 Saint Pierre, Reunion Island, France. Electronic address: isabelle.robene@cirad.fr.
Isabelle
I
Robène
CIRAD, UMR Peuplements Végétaux et Bioagresseurs en Milieu Tropical, CIRAD-Université de la Réunion, Pôle de Protection des Plantes (3P), 7, chemin de l'IRAT, 97410 Saint Pierre, Reunion Island, France.
Marion
M
Perret
CIRAD, UMR Peuplements Végétaux et Bioagresseurs en Milieu Tropical, CIRAD-Université de la Réunion, Pôle de Protection des Plantes (3P), 7, chemin de l'IRAT, 97410 Saint Pierre, Reunion Island, France.
Emmanuel
E
Jouen
CIRAD, UMR Peuplements Végétaux et Bioagresseurs en Milieu Tropical, CIRAD-Université de la Réunion, Pôle de Protection des Plantes (3P), 7, chemin de l'IRAT, 97410 Saint Pierre, Reunion Island, France.
Aline
A
Escalon
CIRAD, UMR Peuplements Végétaux et Bioagresseurs en Milieu Tropical, CIRAD-Université de la Réunion, Pôle de Protection des Plantes (3P), 7, chemin de l'IRAT, 97410 Saint Pierre, Reunion Island, France.
Marie-Véronique
MV
Maillot
ANSES-Plant Health Laboratory, Pôle de Protection des Plantes (3P), 7, chemin de l'IRAT, 97410 Saint Pierre, Reunion Island, France.
Aude
A
Chabirand
ANSES-Plant Health Laboratory, Pôle de Protection des Plantes (3P), 7, chemin de l'IRAT, 97410 Saint Pierre, Reunion Island, France.
Aurélie
A
Moreau
CIRAD, UMR Peuplements Végétaux et Bioagresseurs en Milieu Tropical, CIRAD-Université de la Réunion, Pôle de Protection des Plantes (3P), 7, chemin de l'IRAT, 97410 Saint Pierre, Reunion Island, France.
Annie
A
Laurent
CIRAD, UMR Peuplements Végétaux et Bioagresseurs en Milieu Tropical, CIRAD-Université de la Réunion, Pôle de Protection des Plantes (3P), 7, chemin de l'IRAT, 97410 Saint Pierre, Reunion Island, France.
Frédéric
F
Chiroleu
CIRAD, UMR Peuplements Végétaux et Bioagresseurs en Milieu Tropical, CIRAD-Université de la Réunion, Pôle de Protection des Plantes (3P), 7, chemin de l'IRAT, 97410 Saint Pierre, Reunion Island, France.
Olivier
O
Pruvost
D003429:Cross Reactions; D005189:False Positive Reactions; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D019697:Onions / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012639:Seeds / Q000382:microbiology*; D012680:Sensitivity and Specificity; D053532:Xanthomonas axonopodis / Q000235:genetics / Q000302:isolation & purification*
Diagnostics; Onion seed; Real time quantitative PCR; Xanthomonas