Quantitative Real-time PCR for diagnosis and
identifi-cation of
Xanthomonas citri
pv.
citri
pathotypes,
causal agents of Asiatic Citrus Canker
Asiatic Citrus Canker is induced by
• Xanthomonas citri
pv. citri (Xcc) (EPPO List A1)
Two pathotypes with different economical impacts:
•
X. citri
- pv. citri-A : wide host range of citrus and other related genera, worldwide distribution
X. citri
- pv. citri-A*: narrow host range : limes (C. aurantifolia) and alemow (C. macrophylla),
li-mited areas
X. citri
- pv. citri -Aw : a junior synonym of
X. citri pv. citri -A* (1)
Preliminary study: evaluation of different Xcc
•
primers previously published for PCR diagnosis: Collection of
- Xcc (n=37) and other
Xantho-monas species and pvs. (n=24)
Lack of specificity of PCR-based diagnostic
-tools (included primers proposed in the EPPO standard protocol)
Detection of
X. citri
pv.
citri
and
identification of pathotypes by qPCR
Hydrolysis probes: Taqman-MGB
• Duplex qPCR • Markers • Xcc-Chem: - Xcc specific sequence Xcc-hK:
- Xcc-A specific sequence from a
housekeeping gene
Detection of
Xcc
by PCR in pure
cultures
With three PCR primers designed on genes
•
identified based on from the genome of strain IAPAR 306 (Xcc-A):
involved in pathogenicity (Flm)
related to chemotaxism (Chem)
-Specific of
• X. citri pv. citri
No pathotype specific mutation valuable
•
for qPCR
Perspectives
Development of internal standard Validation of the assay
•
Calibration of the extraction
•
Citrus-18S: a sequence of the 18S gene from Citrus plant
• Sensitivity Detection of 10 • 7 to103 bacteria/ml Detection of 2.5x10 • 6 to 2.5x103 bacteria/mg of lime leaves Specificity
Assays on pure cultures
•
No other
• Xanthomonas
(pathogenic or not on Citrus) amplified with Xcc-Chem system, spe-cific to X. citri pv. citri
Extraction method
Conclusion
Rapid detection of the bacterium in tissues with internal
stan-•
dard
Distinguish pathotypes with different economic incidence
•
Useful for indexing propagation material in nurseries and for
•
surveillance of international movement of X. citri pv. citri
Further assays are necessary to validate the internal standard
•
Delcourt S., Robène-Soustrade I., Vernière C., Boyer C., Pruvost O. CIRAD- UMR Peuplements Végétaux et Bioagresseurs en Milieu Tropical, Saint-Pierre, La Réunion, France
Develop a reliable diagnostic tool to :
Detect• X. citri pv. citri = pathovar specific
Identify both pathotypes = pathotype specific
•
Exclude other Xanthomonads including those
pa-•
thogenic on Citrus: X. citri pv. aurantifolii, X. citri pv.
bilvae, X. alfalfae pv. citrumelonis
100 µl of extract
Wizard® Genomic DNA purification ( Promega)
(1) Bui Thi Ngoc L, et al. (2010) IJSEM vol 60:515-525
0.0 0.2 0.4 0.6 0.8 1.0
Detection of Xanthomonas strains by primers used for Xcc diagnosis
Primers Frequency of detection 2/3 4/7 J−pth J−Rx King VM XAC01/02 XACF/X ACR XCF/XCR Target Non target
40 mg of healthy lime leaves at -80°C crushed with a mortar
+ 108 bacteria Xcc-A (n=21) Non-target Xanthomonas (n=24) Xcc-A* (n=16) Xcc-Chem Xcc-Chem Xcc-Chem Xcc-hK (variable) Xcc-hK
Xcc-hK Pure cultures of Xcc IAPAR 306
Xcc-hK efficacity 81% R2=0.996 Xcc-Chem efficacity 94% R2=0.994
Plant extract contaminated with Xcc IAPAR 306
Xcc-hK efficacity 74% R2=0.997 Xcc-Chem efficacity 87% R2=0.997
This work was funded by the program POSEIDOM interDOM
