Abstract
BackgroundXanthomonads are an important clade of Gram-negative bacteria infecting a plethora of economically important host plants, including citrus. Knowledge about the pathogen’s diversity and population structure are prerequisite for epidemiological surveillance and efficient disease management. Rapidly evolving genetic loci, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), are of special interest to develop new molecular typing tools.ResultsWe analyzed CRISPR loci of 56 Xanthomonas citri pv. citri strains of world-wide origin, a regulated pathogen causing Asiatic citrus canker in several regions of the world. With one exception, 23 unique sequences built up the repertoire of spacers, suggesting that this set of strains originated from a common ancestor that already harbored these 23 spacers. One isolate originating from Pakistan contained a string of 14 additional, probably more recently acquired spacers indicating that this genetic lineage has or had until recently the capacity to acquire new spacers. Comparison of CRISPR arrays with previously obtained molecular typing data, such as amplified fragment length polymorphisms (AFLP), variable-number of tandem-repeats (VNTR) and genome-wide single-nucleotide polymorphisms (SNP), demonstrated that these methods reveal similar evolutionary trajectories. Notably, genome analyses allowed to generate a model for CRISPR array evolution in X. citri pv. citri, which provides a new framework for the genealogy of the citrus canker pathogen.ConclusionsCRISPR-based typing will further improve the accuracy of the genetic identification of X. citri pv. citri outbreak strains in molecular epidemiology analyses, especially when used concomitantly with another genotyping method.
Highlights
Xanthomonads are an important clade of Gram-negative bacteria infecting a plethora of economically important host plants, including citrus
PCR screening of X. citri strains for the presence of the cas1 gene In order to elucidate whether Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas loci are widespread among strains of X. citri pv. citri, we first screened our strain collection (n = 56) as well as a citruspathogenic X. citri pv. bilvae strain for the presence of cas1, the most conserved cas gene, by conventional PCR using cas1-specific primers
A DNA fragment of approximately 220 bp corresponding to the cas1 gene was amplified from all 56 X. citri pv. citri strains (Additional file 1: Figure S1), indicating that these strains may possess a CRISPR/Cas locus of potential use for molecular typing
Summary
Xanthomonads are an important clade of Gram-negative bacteria infecting a plethora of economically important host plants, including citrus. Pathogenic members of the genus cause diseases on over 300 host plants [1]. Citrus canker is commonly used as a generic term that includes two diseases of citrus caused by strains of Xanthomonas citri. South American citrus canker, which is caused by X. citri pv. Citrumelonis, were reported as citrus pathogens but they produce necrotic spots rather than canker-like lesions and are considered minor pathogens [7,8,9,10]. Both canker-causing pathovars were further subdivided into pathotypes (i.e. groups of strains differing in host range within the Citrus genus). Three (A, A* and Aw) and two (B and C) pathotypes are recognized within X. citri pv. citri and X. citri pv. aurantifolii, respectively [11,12,13]
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