Detecting Xanthomonas axonopodis pv. allii
from onion seed by Real Time Multiplex PCR
Bacterial Blight of Onion (BBO), an emergent disease
•
threatening world onion production
Causal agent :
•
Xanthomonas axonopodis pv. allii (Xaa),
a seedtransmitted bacterium listed in quarantine EPPO
A1 list since 2009
Xaa strains belong to
•
X. axonopodis group 9.2
(1)A Multiplex Nested-PCR was previously developed
•
to specifically detect a worldwide collection of Xaa
strains
(2)Multiplex PCR
Specific primer-probe sets targeting two Xaa sequences
•
highly similar with :
portions of contiguous genes
- pilW/pilX in X. euvesica-toria avirulence gene - avrRxv in X. euvesicatoria Internal control •
specific primer-probe set targeting the NADH
dehy
-drogenase gene from Alliaceae
validation of assay for Ct ranging from 22 to 25
-Sensitivity
Standard curves with high correlation coefficients :
•
r2 > 0.99 in the range of 5.103 to 5.107 CFU/g for seed
artifi-cially inoculated with strains containing PILI or AVR marker qPCR efficiency from 80 to 96%
•
Specificity
All strains of Xaa amplified
•
Amplification of a few bacteria from group 9.2 but not
patho-•
genic to onion
Sample processing
Conclusions
Rapid detection and quantification
•
of all known strains of X. a. pv.
allii from onion seed
Development of an
•
internal positive control
in onion seed samples
Amplification from strains non pathogenic to onion.
•
Confirmation can be made by sequence signature of amplicons or
•
isolation on NCTM1 semi-selective medium
(3)followed by
molecu-lar characterization (AFLP
®, MLSA)
Useful tool for
•
global surveillance
and seed health certification
Objectives
Development of a multiplex quantitative Real Time PCR
•
based diagnostic tool to detect specifically X. a. pv allii in
seed lots, in a seed certification prospect
Validation of the assay by the use of a internal control in
•
case of no bacterial DNA amplification
PILI and AVR amplification plots for Xaa concentration ranging from 103 to 107
CFU/mL from artificially inoculated seed
(1) Roumagnac P, Gagnevin L, Gardan L, Sutra L, Manceau C, et al. (2004), Int J Syst Evol Mi-crobiol 54: 15-24
(2) Robene-Soustrade I, Legrand D, Gagnevin L, Chiroleu F, Laurent A, Pruvost O (2010), Appl Environ Microbiol 76(9): 2697-2703
(3) Roumagnac P, Gagnevin L, Pruvost O (2000), Eur J Plant Pathol 106: 867-877
Strains tested
PILI+ and/or AVR+
qPCR-detection
Xaa strains 30/30
X. axonopodis 9.2 group 7/11
other X. axonopodis groups 0/11
other Xanthomonas species 0/4
other phytogenic bacteria 0/4
saprophytic bacteria 0/10
Aline Escalon, Isabelle Robène-Soustrade, Emmanuel Jouen, and Olivier Pruvost UMR « Peuplements Végétaux et Bioagresseurs en Milieu Tropical », CIRAD-Université de la Réunion, France
Real-Time PCR using hydrolysis probe :
Less time consuming
•
Introduction of an internal positive control
•
Can deal with a great number of samples
•
DNA extraction using DNeasy® Plant
mini-kit (Qiagen) 48h at 4°C
in 10mM tris buffer pH 7.2 (10 g seed/50 ml buffer)
mildly crushed and homogenized in a Stomacher® 0 10 20 30 40 −8 −6 −4 −2 0
Multiplex Amplification plot
Cycle (g ol ∆R n) 107 CFU/mL 106 CFU/mL 105 CFU/mL 104 CFU/mL 103 CFU/mL Internal control avr pili ● ● ● ● ● ● ● ● ● ●
Standard Curve for Xaa detection
Log Co Ct 3 4 5 6 7 23 27 31 35 slope = 3.41 Efficiency=96.29% R2=0.997
