Molecular Epidemiology of Ralstonia pseudosolanacearum Phylotype I Strains in the Southwest Indian Ocean Region and Their Relatedness to African Strains.

The increasing requirement for developing tools enabling fine strain traceability responsible for epidemics is tightly linked with the need to understand factors shaping pathogen populations and their environmental interactions. Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is one of the most important plant diseases in tropical and subtropical regions. Sadly, little, outdated, or no information on its epidemiology is reported in the literature, although alarming outbreaks are regularly reported as disasters. A large set of phylotype I isolates (n = 2,608) was retrieved from diseased plants in fields across the Southwest Indian Ocean (SWIO) and Africa. This collection enabled further assessment of the epidemiological discriminating power of the previously published RS1-MLVA14 scheme. Thirteen markers were validated and characterized as not equally informative. Most had little infra-sequevar polymorphism, and their performance depended on the sequevar. Strong correlation was found with a previous multilocus sequence typing scheme. However, 2 to 3% of sequevars were not correctly assigned through endoglucanase gene sequence. Discriminant analysis of principal components (DAPC) revealed four groups with strong phylogenetic relatedness to sequevars 31, 33, and 18. Phylotype I-31 isolates were highly prevalent in the SWIO and Africa, but their dissemination pathways remain unclear. Tanzania and Mauritius showed the greatest diversity of RSSC strains, as the four DAPC groups were retrieved. Mauritius was the sole territory harboring a vast phylogenetic diversity and all DAPC groups. More research is still needed to understand the high prevalence of phylotype I-31 at such a large geographic scale.

Contrasting genetic diversity and structure among Malagasy Ralstonia pseudosolanacearum phylotype I populations inferred from an optimized Multilocus Variable Number of Tandem Repeat Analysis scheme.

The Ralstonia solanacearum species complex (RSSC), composed of three species and four phylotypes, are globally distributed soil-borne bacteria with a very broad host range. In 2009, a devastating potato bacterial wilt outbreak was declared in the central highlands of Madagascar, which reduced the production of vegetable crops including potato, eggplant, tomato and pepper. A molecular epidemiology study of Malagasy RSSC strains carried out between 2013 and 2017 identified R. pseudosolanacearum (phylotypes I and III) and R. solanacearum (phylotype II). A previously published population biology analysis of phylotypes II and III using two MultiLocus Variable Number of Tandem Repeats Analysis (MLVA) schemes revealed an emergent epidemic phylotype II (sequevar 1) group and endemic phylotype III isolates. We developed an optimized MLVA scheme (RS1-MLVA14) to characterize phylotype I strains in Madagascar to understand their genetic diversity and structure. The collection included isolates from 16 fields of different Solanaceae species sampled in Analamanga and Itasy regions (highlands) in 2013 (123 strains) and in Atsinanana region (lowlands) in 2006 (25 strains). Thirty-one haplotypes were identified, two of them being particularly prevalent: MT007 (30.14%) and MT004 (16.44%) (sequevar 18). Genetic diversity analysis revealed a significant contrasting level of diversity according to elevation and sampling region. More diverse at low altitude than at high altitude, the Malagasy phylotype I isolates were structured in two clusters, probably resulting from different historical introductions. Interestingly, the most prevalent Malagasy phylotype I isolates were genetically distant from regional and worldwide isolates. In this work, we demonstrated that the RS1-MLVA14 scheme can resolve differences from regional to field scales and is thus suited for deciphering the epidemiology of phylotype I populations.

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

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.

Transcriptome analysis revealed that a quorum sensing system regulates the transfer of the pAt megaplasmid in Agrobacterium tumefaciens.

BACKGROUND: Agrobacterium tumefaciens strain P4 is atypical, as the strain is not pathogenic and produces a for this species unusual quorum sensing signal, identified as N-(3-hydroxy-octanoyl)-homoserine lactone (3OH,C8-HSL). RESULTS: By sequence analysis and cloning, a functional luxI-like gene, named cinI, has been identified on the At plasmid of A. tumefaciens strain P4. Insertion mutagenesis in the cinI gene and transcriptome analyses permitted the identification of 32 cinI-regulated genes in this strain, most of them encoding proteins responsible for the conjugative transfer of pAtP4. Among these genes were the avhB genes that encode a type 4 secretion system (T4SS) involved in the formation of the conjugation apparatus, the tra genes that encode the DNA transfer and replication (Dtr) machinery and cinI and two luxR orthologs. These last two genes, cinR and cinX, exhibit an unusual organization, with the cinI gene surrounded by the two luxR orthologs. Conjugation experiments confirmed that the conjugative transfer of pAtP4 is regulated by 3OH,C8-HSL. Root colonization experiments indicated that the quorum sensing regulation of the conjugation of the pAtP4 does not confer a gain or a loss of fitness to the bacterial host in the tomato plant rhizosphere. CONCLUSION: This work is the first identification of the occurrence of a quorum sensing regulation of the pAt conjugation phenomenon in Agrobacterium.