Identification of Xanthomonas fragariae, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas fuscans subsp. fuscans with Novel Markers and Using a Dot Blot Platform Coupled with Automatic Data Analysis

Abstract

Phytosanitary regulations and the provision of plant health certificates still rely mainly on long and laborious culture-based methods of diagnosis, which are frequently inconclusive. DNA-based methods of detection can circumvent many of the limitations of currently used screening methods, allowing a fast and accurate monitoring of samples. The genus Xanthomonas includes 13 phytopathogenic quarantine organisms for which improved methods of diagnosis are needed. In this work, we propose 21 new Xanthomonas-specific molecular markers, within loci coding for Xanthomonas-specific protein domains, useful for DNA-based methods of identification of xanthomonads. The specificity of these markers was assessed by a dot blot hybridization array using 23 non-Xanthomonas species, mostly soil dwelling and/or phytopathogens for the same host plants. In addition, the validation of these markers on 15 Xanthomonas spp. suggested species-specific hybridization patterns, which allowed discrimination among the different Xanthomonas species. Having in mind that DNAbased methods of diagnosis are particularly hampered for unsequenced species, namely, Xanthomonas fragariae, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas fuscans subsp. fuscans, for which comparative genomics tools to search for DNA signatures are not yet applicable, emphasis was given to the selection of informative markers able to identify X. fragariae, X. axonopodis pv. phaseoli, and X. fuscans subsp. fuscans strains. In order to avoid inconsistencies due to operator-dependent interpretation of dot blot data, an image-processing algorithm was developed to analyze automatically the dot blot patterns. Ultimately, the proposed markers and the dot blot platform, coupled with automatic data analyses, have the potential to foster a thorough monitoring of phytopathogenic xanthomonads.Xanthomonas is a genus of Gammaproteobacteria that includes numerous phytopathogenic species, each characterized by a narrow host range. However, as a whole, the genus members are able to infect a broad range of plants, distributed over 124 monocotyledonous and 268 dicotyledonous plant species (15). The nomenclature of this complex genus is still under debate, and the taxonomic rank of many previously described pathovars has been revised (28,41,48). At the moment, the European and Mediterranean Plant Protection Organization (EPPO) recommends that 13 members of the genus Xanthomonas be considered quarantine pests. Therefore, reliable, fast, and technically and commercially accessible screening methods of detection and identification are needed to allow the survey of a large number of samples. This would ensure the phytosanitary certification of plants, prevent the spread of contaminated plant material, and facilitate the implementation of timely phytosanitation and quarantine measures (4).The current certified methods of bacterial detection rely mainly on culture-based approaches and plant bioassays (35). While these methods allow for a presumptive identification, they lack resolution of dete...

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.…”

A multiplex-PCR assay for identification of the quarantine plant pathogen Xanthomonas axonopodis pv. phaseoli

“…When screening for adapted markers, collections of strains used should span over the full diversity of the 4 genetic lineages of the pathovar phaseoli. In the recent work by Albuquerque et al (2011), identification assays by dot-blot were developed for the identification of strains belonging to various pathovars of X. axonopodis. However it is not clear whether strains representing the diversity of each of the 4 genetic lineages of the pathovar phaseoli were used to screen for markers.…”

“…The latter sets of primers thus cannot be used for routine identification of Xap on seed lots. Recently Albuquerque et al (2011) reported the development of a dot-blot assay for the identification of Xap. However dot-blot analysis is much time consuming, and large numbers of samples are difficult to perform when no access to dedicated platforms is available.…”

Detection and quantification of Xanthomonas axonopodis pv. phaseoli and its variant fuscans in common bean seeds

“…The common bacterial blight of bean (CBCF), incited by Xanthomonas axonopodis pv. phaseoli (Xap) (Smith) Vauterin et al (1995) and by its variant fuscans (Xapf), described by Burkholder and Bullard (1946), is characterized as a disseminated disease in most regions that produce beans (Phaseolus vulgaris L.) in the world (Albuquerque et al, 2011;Alavi et al, 2008;Saettler, 1991;Gilbertson, 1994;Sartorato and Rava, 1994;Bianchini et al, 2005) being considered as quarantine pathogen in countries such as the United States, Croatia and countries of the European and Mediterranean Organization for Plant Protection (EPPO).…”