A duplex quantitative real-time PCR assay for the detection and quantification of Xanthomonas phaseoli pv. dieffenbachiae from diseased and latently infected anthurium tissue

• Published in: Journal of microbiological methods 2019-06, Vol.161, p.74-83
• Main Authors: Jouen, Emmanuel, Chiroleu, Fréderic, Maillot-Lebon, Véronique, Chabirand, Aude, Merion, Sabine, Boyer, Claudine, Pruvost, Olivier, Robène, Isabelle
• Format: Article
• Language: English
• Subjects: Anthurium; Diagnostics; Latent infections; Real-time quantitative PCR; Xanthomonas phaseoli pv. dieffenbachiae
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2019.03.003

Anthurium bacterial blight caused by Xanthomonas phaseoli pv. dieffenbachiae (formerly Xanthomonas axonopodis pv. dieffenbachiae) is the major phytosanitary threat in many anthurium growing areas worldwide. Reliable and sensitive diagnostic tools are required for surveillance and certification programs. A duplex real–time quantitative PCR assay was developed for the detection and quantification of X. phaseoli pv. dieffenbachiae from anthurium tissue. This PCR assay targeted a X. phaseoli pv. dieffenbachiae–specific gene encoding an ABC transporter and an internal control encoding for chalcone synthase in Anthurium andreanum. A cycle threshold (Ct), using a receiver-operating characteristic approach (ROC), was implemented to ensure that the declaration of a positive sample was reliable. The duplex real–time assay displayed very high performance with regards to analytical specificity (100% inclusivity, 98.9% exclusivity), analytical sensitivity (LOD95% = 894 bacteria/ml corresponding to 18 bacteria per reaction) and repeatability. We demonstrated the pertinence of this real–time quantitative PCR assay for detecting X. phaseoli pv. dieffenbachiae from diseased leaf tissue (collected from outbreaks on anthurium) and from asymptomatic, latently infected anthurium plants. This assay could be useful for surveillance, as well as for indexing propagative plant material for the presence of X. phaseoli pv. dieffenbachiae. •We developed a qPCR for detecting X. phaseoli pv. dieffenbachiae from diseased or latently infected anthurium leaves.•The assay includes an internal amplification control targeting anthurium tissue.•A cycle cut-off value was implemented to ensure that the declaration of a positive sample was reliable.•The limit of detection was estimated at 18 bacteria per reaction.•This molecular tool could be useful for surveillance and for the production of healthy propagative plant material.