Molecular Epidemiology of Bacterial Wilt in the Madagascar Highlands Caused by Andean (Phylotype IIB-1) and African (Phylotype III) Brown Rot Strains of the Ralstonia solanacearum Species Complex

Abstract

The Ralstonia solanacearum species complex (RSSC) is a highly diverse cluster of bacterial strains found worldwide, many of which are destructive and cause bacterial wilt (BW) in a wide range of host plants. In 2009, potato production in Madagascar was dramatically affected by several BW epidemics. Controlling this disease is critical for Malagasy potato producers. The first important step toward control is the characterization of strains and their putative origins. The genetic diversity and population structure of the RSSC were investigated in the major potato production areas of the Highlands. A large collection of strains (n = 1224) was assigned to RSSC phylotypes based on multiplex polymerase chain reaction (PCR). Phylotypes I and III have been present in Madagascar for a long time but rarely associated with major potato BW outbreaks. The marked increase of BW prevalence was found associated with phylotype IIB sequevar 1 (IIB-1) strains (n = 879). This is the first report of phylotype IIB-1 strains in Madagascar. In addition to reference strains, epidemic IIB-1 strains (n = 255) were genotyped using the existing MultiLocus Variable-Number Tandem Repeat Analysis (MLVA) scheme RS2-MLVA9, producing 31 haplotypes separated into two related clonal complexes (CCs). One major CC included most of the worldwide haplotypes distributed across wide areas. A regional-scale investigation suggested that phylotype IIB-1 strains were introduced and massively spread via latently infected potato seed tubers. Additionally, the genetic structure of phylotype IIB-1 likely resulted from a bottleneck/founder effect. The population structure of phylotype III, described here for the first time in Madagascar, exhibited a different pattern. Phylotype III strains (n = 217) were genotyped using the highly discriminatory MLVA scheme RS3-MLVA16. High genetic diversity was uncovered, with 117 haplotypes grouped into 11 CCs. Malagasy phylotype III strains were highly differentiated from continental African strains, suggesting no recent migration from the continent. Overall, population structure of phylotype III involves individual small CCs that correlate to restricted geographic areas in Madagascar. The evidence suggests, if at all, that African phylotype III strains are not efficiently transmitted through latently infected potato seed tubers.

Keywords: MLVA; Madagascar; Ralstonia solanacearum; genetic diversity; population structure.

FIGURE 1

Spatial distribution and frequency of the phylotypes I, II, and III across the 12 AEZs in the Central Highlands of Madagascar.

FIGURE 2

Minimum spanning tree (MST) representation of the MLVA clustering. (A) MST of global RSSC phylotype IIB-1 strains (n = 285). (B) MST of Malagasy RSSC phylotype IIB-1 strains collected in the Central Highlands of Madagascar (n = 255). (C) MST of African RSSC phylotype III strains (n = 280). (D) MST of Malagasy RSSC phylotype III strains collected in the Central Highlands of Madagascar (n = 217). Each circle represents a unique MLVA haplotype (MT) and its size is proportional to the number of strains having the same MT. The thicker branches link MT differing by only one TR locus (SLV), the thinner branches link MT that differ in 2 TR loci (DLV), and the gray dashed branches link MT that differ in three TR loci. No link is indicative of variation at >4 loci. Color represents the country or the agroecological zone of origin. Halos indicate the distinct clonal complexes (CCs). CSC, clonal sub-complex.

FIGURE 3

GENELAND analysis of RSSC populations in Central Highlands of Madagascar. (A) Spatially explicit estimate of population clusters, and the distribution of each phylotype IIB-1 genetic cluster defined by GENELAND at K = 3 from 70% posterior probability assuming uncorrelated allele frequency model (n = 255). The highest membership values are in light yellow, and the curves indicate spatial changes in assignment values. (B) Spatial distribution and population structure of the RSSC phylotype III strains inferred in GENELAND analyses at K = 11 from 70% posterior probability assuming correlated allele frequency (n = 217). The black dots indicate the sampling locations. The plot is based on the highest probability run for a given value of K. The abscissa and ordinate show the coordinates of sampling locations. (C) General flow of potato seed system in the potato growing basins.

FIGURE 4

Minimum spanning tree (MST) representation of RSSC phylotype IIB-1 strains during the survey period. (A) MST of 255 phylotype IIB-1 strains. (B) MST of 206 phylotype IIB-1 strains collected in April 2013. (C) MST of 49 phylotype IIB-1 strains collected in December 2013. Each circle represents a unique MLVA haplotype (MT), and its size is proportional to the number of strains with the same MT. Color represents the population clusters computed in GENELAND. (D) Distribution of the sampling sites during the two survey sampling periods, April and December 2013.