Genetic Diversity of the Ralstonia solanacearum Species Complex in the Southwest Indian Ocean Islands

Abstract

Epidemiological surveillance of plant pathogens based on genotyping methods is mandatory to improve disease management strategies. In the Southwest Indian Ocean (SWIO) islands, bacterial wilt (BW) caused by the Ralstonia solanacearum species complex (RSSC) is hampering the production of many sustainable and cash crops. To thoroughly analyze the genetic diversity of the RSSC in the SWIO, we performed a wide sampling survey (in Comoros, Mauritius, Reunion, Rodrigues, and Seychelles) that yielded 1,704 isolates from 129 plots, mainly from solanaceous crops. Classification of the isolates to the four major RSSC phylogenetic groups, named phylotypes, showed that 87% were phylotype I, representing the most prevalent strain in each of the SWIO islands. Additionally, 9.7% were phylotype II, and 3.3% were phylotype III; however, these isolates were found only in Reunion. Phylotype IV (2 isolates), known to be restricted to Indonesia-Australia-Japan, was reported in Mauritius, representing the first report of this group in the SWIO. Partial endoglucanase (egl) sequencing, based on the selection of 145 isolates covering the geographic and host diversity in the SWIO (also including strains from Mayotte and Madagascar), revealed 14 sequevars with Reunion and Mauritius displaying the highest sequevar diversity. Through a multilocus sequence analysis (MLSA) scheme based on the partial sequencing of 6 housekeeping genes (gdhA, gyrB, rplB, leuS, adk, and mutS) and 1 virulence-associated gene (egl), we inferred the phylogenetic relationships between these 145 SWIO isolates and 90 worldwide RSSC reference strains. Phylotype I was the most recombinogenic, although recombination events were detected among all phylotypes. A multilocus sequence typing (MLST) scheme identified 29 sequence types (STs) with variable geographic distributions in the SWIO. The outstanding epidemiologic feature was STI-13 (sequevar I-31), which was overrepresented in the SWIO and obviously reflected a lineage strongly adapted to the SWIO environment. A goeBURST analysis identified eight clonal complexes (CCs) including SWIO isolates, four CCs being geographically restricted to the SWIO, and four CCs being widespread beyond the SWIO. This work, which highlights notable genetic links between African and SWIO strains, provides a basis for the epidemiological surveillance of RSSC and will contribute to BW management in the SWIO.

Keywords: MLSA; MLST; Ralstonia solanacearum species complex; Southwest Indian Ocean; clonal complex; epidemiology; genetic diversity.

Figure 1

Distribution of sequevars of the RSSC depending on altitude and host of isolation in SWIO islands. For each sequevar, the dots correspond to the different altitudes of isolation. On the right of the dots, the letters indicate the host of isolation, and the rectangles mention the range of altitude of isolation for each plant.

Figure 2

Phylogenetic tree based on the concatenated seven genes (gdhA, gyrB, rplB, leuS, adk, mutS, and egl). Supports of branch were generated by aLRT. Red branches show aLRT support value >0.5, orange between (0.2 ≤ × ≤ 0.5), green <0.2.

Figure 3

Minimum spanning tree (MST) from MLST data displaying genetic relationships between RSSC SWIO and worldwide reference strains (C3 collection, n = 235). MLST data are based on seven gene regions (gdhA, mutS, adk, leuS, rplB, gyrB, and egl). The dot colors indicate the country of isolation. Worldwide reference strains: Africa (brown), America (green), Asia (yellow), Oceania (black), Europe (white). SWIO strains: Reunion (red), Maurice (blue metallic), Comoros (pink), Madagascar (purple), Seychelles (turquoise), Rodrigues (sky blue) and Mayotte (orange). The labels in the dots indicate the MLST sequence type numbers (ST). Black thick lines, black regular lines, gray thin lines, and gray dashed lines joining haplotypes indicate single, double, triple, and more variations, respectively. No link is indicative of variations at >3 loci. Red halos denote a clonal complex (CC).