Quantitative Real-time PCR for diagnosis and identifi ...
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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 to10 3 bacteria/ml Detection of 2.5x10 • 6 to 2.5x10 3 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/XACR XCF/XCR T arget Non target 40 mg of healthy lime leaves at -80°C crushed with a mortar + 10 8 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% R 2 =0.996 Xcc-Chem efficacity 94% R 2 =0.994 Plant extract contaminated with Xcc IAPAR 306 Xcc-hK efficacity 74% R 2 =0.997 Xcc-Chem efficacity 87% R 2 =0.997 This work was funded by the program POSEIDOM interDOM Citrus-18S
Transcript of Quantitative Real-time PCR for diagnosis and identifi ...
Quantitative Real-time PCR for diagnosis and identifi-cation of Xanthomonas citri pv. citri pathotypes,
causal agents of Asiatic Citrus CankerAsiatic 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 areasX. 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 sequenceXcc-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
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0.8
1.0
Detection of Xanthomonas strains by primers used for Xcc diagnosis
Primers
Freq
uenc
y of
det
ecti
on
2/3 4/7J−p
thJ−R
xKing
VM
XAC01/02
XACF/XACR
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-hKPure cultures of Xcc IAPAR 306
Xcc-hK efficacity 81% R2=0.996
Xcc-Chemefficacity 94%R2=0.994
Plant extract contaminated with Xcc IAPAR 306
Xcc-hKefficacity 74%R2=0.997
Xcc-Chemefficacity 87%R2=0.997
This work was funded by the program POSEIDOM interDOM
Citrus-18S
