Evolutionary and Experimental Assessment of Novel Markers for Detection of Xanthomonas euvesicatoria in Plant Samples

Abstract

BackgroundBacterial spot-causing xanthomonads (BSX) are quarantine phytopathogenic bacteria responsible for heavy losses in tomato and pepper production. Despite the research on improved plant spraying methods and resistant cultivars, the use of healthy plant material is still considered as the most effective bacterial spot control measure. Therefore, rapid and efficient detection methods are crucial for an early detection of these phytopathogens.MethodologyIn this work, we selected and validated novel DNA markers for reliable detection of the BSX Xanthomonas euvesicatoria (Xeu). Xeu-specific DNA regions were selected using two online applications, CUPID and Insignia. Furthermore, to facilitate the selection of putative DNA markers, a customized C program was designed to retrieve the regions outputted by both databases. The in silico validation was further extended in order to provide an insight on the origin of these Xeu-specific regions by assessing chromosomal location, GC content, codon usage and synteny analyses. Primer-pairs were designed for amplification of those regions and the PCR validation assays showed that most primers allowed for positive amplification with different Xeu strains. The obtained amplicons were labeled and used as probes in dot blot assays, which allowed testing the probes against a collection of 12 non-BSX Xanthomonas and 23 other phytopathogenic bacteria. These assays confirmed the specificity of the selected DNA markers. Finally, we designed and tested a duplex PCR assay and an inverted dot blot platform for culture-independent detection of Xeu in infected plants.SignificanceThis study details a selection strategy able to provide a large number of Xeu-specific DNA markers. As demonstrated, the selected markers can detect Xeu in infected plants both by PCR and by hybridization-based assays coupled with automatic data analysis. Furthermore, this work is a contribution to implement more efficient DNA-based methods of bacterial diagnostics.

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

“…For molecular-based pathogen diagnose, the selected DNA signatures must be highly specific for the target pathogen to provide reliable detection results. By the continuously increasing amount of genome sequences, new unique targets were screened thorough comparative genomic analysis carried out in silico, and validated using PCR and/or hybridization-based approaches (Mazumder et al 2005;Phillippy et al 2007;Albuquerque et al 2011Albuquerque et al , 2012Bühlmann et al 2013;Ash et al 2014). Although no draft genome sequence of X. axonopodis pv.…”

Molecular Epidemiology ofXanthomonas perforansOutbreaks in Tomato Plants from Transplant to Field as Determined by Single-Nucleotide Polymorphism Analysis

“…MLSA and MLVA are commonly used tools to characterize bacterial spot of tomato (BST) outbreaks and epidemics caused by X. perforans worldwide (19)(20)(21)(22)(23)(24)(25)(26)(27)(28). MLSA-based analysis of X. perforans revealed two subgroups in a global collection of strains (28).…”

A quantitative hybridization approach using 17 DNA markers for identification and clustering analysis of Ralstonia solanacearum

“…Thus, an identification and typing procedure can be performed by digoxigenin-labelling DNA from a new isolate and carrying out hybridization against all 17 markers. This should allow the quantification of the hybridization signals for all markers simultaneously, by using an inverted dot blot approach coupled with automatic analysis of hybridization data (Albuquerque et al, 2012); then, the new isolate's position within the dendrogram can be ascertained to infer its typing profile.…”

“…1). As previously suggested, the inclusion of comparative genomics in marker selection workflows constitutes a powerful tool to assess the specificity and infer the stability of each marker (Albuquerque et al, 2012). The major drivers of gene loss/gain in Rs are still being investigated (Fall et al, 2007;Guidot et al, 2009a) and, for some of the analysed regions, the exact mechanisms of this gene gain/loss in Ralstonia do not appear to be linked to typical mobilization-related features.…”