A MLVA genotyping scheme for global surveillance of the citrus pathogen Xanthomonas citri pv. citri suggests a worldwide geographical expansion of a single genetic lineage

Abstract

MultiLocus Variable number of tandem repeat Analysis (MLVA) has been extensively used to examine epidemiological and evolutionary issues on monomorphic human pathogenic bacteria, but not on bacterial plant pathogens of agricultural importance albeit such tools would improve our understanding of their epidemiology, as well as of the history of epidemics on a global scale. Xanthomonas citri pv. citri is a quarantine organism in several countries and a major threat for the citrus industry worldwide. We screened the genomes of Xanthomonas citri pv. citri strain IAPAR 306 and of phylogenetically related xanthomonads for tandem repeats. From these in silico data, an optimized MLVA scheme was developed to assess the global diversity of this monomorphic bacterium. Thirty-one minisatellite loci (MLVA-31) were selected to assess the genetic structure of 129 strains representative of the worldwide pathological and genetic diversity of X. citri pv. citri. Based on Discriminant Analysis of Principal Components (DAPC), four pathotype-specific clusters were defined. DAPC cluster 1 comprised strains that were implicated in the major geographical expansion of X. citri pv. citri during the 20th century. A subset of 12 loci (MLVA-12) resolved 89% of the total diversity and matched the genetic structure revealed by MLVA-31. MLVA-12 is proposed for routine epidemiological identification of X. citri pv. citri, whereas MLVA-31 is proposed for phylogenetic and population genetics studies. MLVA-31 represents an opportunity for international X. citri pv. citri genotyping and data sharing. The MLVA-31 data generated in this study was deposited in the Xanthomonas citri genotyping database (http://www.biopred.net/MLVA/).

Figure 1. Categorical minimum spanning tree from MLVA-31 data (129 strains –72 haplotypes) representing the genetic diversity within a worldwide strain collection of Xanthomonas citri pv. citri in relation with its pathological diversity.

Dot diameter and color are representative of the number of strains per haplotype and pathotype, respectively (red: pathotype A; blue: pathotype A*; green: pathotype A^w). Numbers in dots are for haplotype numbers. Numbers along the links indicate the number of polymorphic TR loci distinguishing haplotypes. Haplotypes in a same colored ellipse were assigned to a same genetic cluster by Discriminant Analysis of Principal Components. Dashed ellipses indicate subclusters, as defined by goeBURST .

Figure 2. Plot describing the discriminatory power (expressed as G/N, the ratio between the number of haplotypes and the number of strains) in relation to the number of TR loci assayed.

Black dashes represent the range of G/N ratios. Red dashes indicate 2.5 and 97.5% quantiles. Red dots indicate the median G/N values.