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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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