Comparative and collaborative studies for the validation of a nested PCR for the detection of Xanthomonas axonopodis pv. dieffenbachiae from Anthurium samples

Abstract

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires sensitive and reliable diagnostic tools. The European standard EN ISO 16140:2003 has been followed to compare a nested PCR assay (N-PCR) to a reference method (isolation and serological identification of bacterial colonies) and to other alternative serological detection methods. The evaluation was performed in two steps: a comparative study and a collaborative study involving 15 European laboratories. Although inclusivity was maximal (100%) for all methods, a maximal exclusivity was obtained only with N-PCR followed by an enzymatic restriction digestion of the amplicons. Exclusivity indices of 90Á6, 88Á7 and 47Á2% were found for indirect ELISA, immunofluorescence and double antibody sandwich ELISA, respectively. An exclusivity of 92Á5% was obtained with the reference method, further increased to 100% if pathogenicity tests were performed as a supplemental assay. The best level of sensitivity (relative detection level) was obtained with the reference method followed by the N-PCR assay. The N-PCR performance in terms of relative accuracy, accordance and concordance was very similar to that of the reference method. Moreover, N-PCR had undeniable advantages compared to the reference method (less labour-intensive and less time-consuming). In addition, post-test probabilities of infection were calculated to select the most appropriate detection scheme related to the prevalence of the pathogen. The N-PCR assay has since been included in a revised version of the EPPO detection protocol.

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of bacterial blight pathogen (Xanthomonas axonopodis pv. dieffenbachiae) in anthurium

“…2009). The developed Nested-PCR method targeting this gene, particularly in terms of specificity, has been confirmed though comparative and collaborative study involving 15 European laboratories, since added in a revised version of the EPPO detection protocol (Chabirand et al 2014). So this fragment maybe represent a Xad-specific gene and suitable for elaboration of molecular detection tool.…”

“…Over the past two decades, various traditional and molecular methods have been developed for Xad characterization, including symptoms recognition, semiselective culture (Norman and Alvarez 1994;Laurent et al 2009), pathogenicity tests, biochemical analysis (Lipp et al 1992;Norman and Alvarez 1994), 16S rDNA sequencing and PCR-based molecular identification (Khoodoo et al 2005a, b;Robène-Soustrade et al 2006;Chabirand et al 2014). Especially the Nested-PCR molecular marker, showed a widely validated Xad-specificity and could be carried out for strains detection from plants (Robène-Soustrade et al 2006;Chabirand et al 2014). However, one or more drawbacks of time-consuming, costliness, dependency of equipments and technology, prevented the application of these procedures in routine or field surveys.…”

Consequences of the taxonomic revision of Xanthomonas axonopodis pv. dieffenbachiae and of the analysis of its associated pathogenicity for the recommendation of regulation (EPPO A2 List)

“…The present PM 7/23 (2) EPPO Diagnostic Standard for X. axonopodis pv. dieffenbachiae (EPPO, 2009) is based on a nested-PCR (N-PCR) method and was recently validated (Chabirand et al, 2014). Since Anthurium is the most economically valued host, the N-PCR method was specifically directed towards identification of those X. axonopodis pv.…”

Field and laboratory screening of anthurium cultivars for resistance to foliar bacterial blight and the induced activities of defence-related enzymes

“…dieffenbachiae (Xad) has been the most devastating disease of anthurium (Anthurium andreanum Hort.) (European and Mediterranean Plant Protection Organization, 2009;Chabirand et al, 2014). This disease is prevalent in commercial anthurium nurseries throughout the world and has caused extensive losses to anthurium industries (European and Mediterranean Plant Protection Organization, 2009).…”