Comparative Genomics of Pathogenic and Nonpathogenic Strains of Xanthomonas arboricola Unveil Molecular and Evolutionary Events Linked to Pathoadaptation

Abstract

The bacterial species Xanthomonas arboricola contains plant pathogenic and nonpathogenic strains. It includes the pathogen X. arboricola pv. juglandis, causing the bacterial blight of Juglans regia. The emergence of a new bacterial disease of J. regia in France called vertical oozing canker (VOC) was previously described and the causal agent was identified as a distinct genetic lineage within the pathovar juglandis. Symptoms on walnut leaves and fruits are similar to those of a bacterial blight but VOC includes also cankers on trunk and branches. In this work, we used comparative genomics and physiological tests to detect differences between four X. arboricola strains isolated from walnut tree: strain CFBP 2528 causing walnut blight (WB), strain CFBP 7179 causing VOC and two nonpathogenic strains, CFBP 7634 and CFBP 7651, isolated from healthy walnut buds. Whole genome sequence comparisons revealed that pathogenic strains possess a larger and wider range of mobile genetic elements than nonpathogenic strains. One pathogenic strain, CFBP 7179, possessed a specific integrative and conjugative element (ICE) of 95 kb encoding genes involved in copper resistance, transport and regulation. The type three effector repertoire was larger in pathogenic strains than in nonpathogenic strains. Moreover, CFBP 7634 strain lacked the type three secretion system encoding genes. The flagellar system appeared incomplete and nonfunctional in the pathogenic strain CFBP 2528. Differential sets of chemoreceptor and different repertoires of genes coding adhesins were identified between pathogenic and nonpathogenic strains. Besides these differences, some strain-specific differences were also observed. Altogether, this study provides valuable insights to highlight the mechanisms involved in ecology, environment perception, plant adhesion and interaction, leading to the emergence of new strains in a dynamic environment.

Keywords: ICE; Juglans regia; bacterial blight; copper resistance; vertical oozing canker.

Figure 1

Venn diagrams illustrating the comparisons of Xanthomonas arboricola genomes.

Figure 2

Circular representation with CGView tool of the orthoMCL analysis of the four X. arboricola genomes. Genomic sequences were compared against each other and BLAST results were converted in a graphical map showing the entire sequences. From inside to outside, circle 1 indicates the G+C content, circles 2 to 5 represent CFBP 7634, CFBP 7651, CFBP 7179, CFBP 2528 locus-tags. The external circle shows COG classification of CDSs with different colors according to the legend. In circles 2 to 5, the color indicates the BLAST score (see legend).

Figure 3

CVTree obtained from X. arboricola genome sequences available in public database. Xanthomonas campestris pv campestris (ATCC 33913); Xanthomonas arboricola pv celebensis (NCPPB 1832, NCPPB 1630); Xanthomonas arboricola (CFBP 7651, CFBP 7634); Xanthomonas arboricola pv juglandis (NCPPB 1447, CFBP 2528, CFBP 7179); Xanthomonas arboricola pv pruni (Xap 33, MAFF 311562, MAFF 301427, MAFF 301420); Xanthomonas arboricola pv corylina (NCCPB 100457). Branch length represents the distance calculated using alignment free composition vector method.

Figure 4

Detection of pectate lyase activity (A) and pectin methyl esterase activity (B) from the strains CFBP 2528 (28), CFBP 7179 (79), CFBP 7651 (51), CFBP 7634 (34). The translucent halo (A) and slightly violet (B) surrounding the colony indicates the activity.

Figure 5

Schematic representation of the T3SS locus of the four X. arboricola strains.

Figure 6

Schematic representation of the flagellar gene cluster of the four X. arboricola strains.

Figure 7

Motility of the four X. arboricola strains. (A) CFBP 2528; (B) CFBP 7179; (C) CFBP 7651; (D) CFBP 7634, 60 h after inoculation in TSA 10% (upper line) or MOKA (lower line), with agar 0.2%.

Figure 8

Multiple alignment between the flagellin FliC N-terminal regions of the 4 X. arboricola strains and the flg22 peptide. The “D” to “V” switch between nonpathogenic and pathogenic strains is shown with the red box.

Figure 9

Schematic representation of T4SS genes of the four X. arboricola strains.

Figure 10

Schematic representation highlighting conserved and differential features between the four genome sequences. Specific features are written using the color code: red for CFBP 7179; orange for CFBP 2528; dark green for CFBP 7651, light green for CFBP 7634.