Epidemiological surveillance of Ralstonia solanacearum, causal agent of bacterial wilt of solanaceous crops, in the South-West Indian Ocean islands and Eastern Africa: diversity and genetic structure of populations

Description

A major initial effort will be required for sampling and collecting epidemiological and historical strains of R. solanacearum strains present on vegetable range of host crops (potato, tomato, eggplant...) across the SWIO islands (Réunion, Comoros, Mauritius and Seychelles) and Eastern Africa (Tanzania, Kenya). This step constitutes the cornerstone of the project and will be conducted through a statistical approach using a georeferenced sampling scheme (GPS). Sampled strains will complement the existing R. solanacearum collection from Mayotte (155 strains collected in 2012) and Madagascar (over 2500 strains collected in 2013) that are being analyzed in the context of S. Ravelomanantsoa’s PhD thesis (2013-2016) within a collaboration between UMR PVBMT and FIFAMANOR.

These strains will then be subjected to a thorough molecular characterization. First, determination of phylotype by multiplex PCR and of sequevar by endoglucanase (egl) gene sequencing will be conducted (Fegan and Prior, 2005). Second, genetic diversity within the R. solanacearum strains will be analyzed by MLSA/MLST. The approach will establish phylogenetic relationships among strains of R. solanacearum originated from SWIO islands and Eastern Africa and their position relative to worldwide reference strains already typed (Wicker et al., 2012). Furthermore, a MLVA scheme targeting tandem repeats with a rapid evolution rate will be used in order to decipher the evolutionary forces acting at the level of populations and therefore their structure. Several scales of analysis will be considered: country, island, region, and field. Spatial analyses will be performed at those different scales. Temporal analyses will be carried out on collections from Reunion to evaluate the level of clonality of R. solanacearum. Population genetics and molecular epidemiology analyses tools will be used to describe the intra- and inter- genetic diversity of these populations and the relationships between the individuals. Pathways of introduction and spread will be inferred using Bayesian scenario choice analyses using the genetic proximity of collected strains against an international reference collection, owned by our team in Reunion (INRA-CIRAD collection “RUN”), comprising over 3200 entries. RUN collection covers all phylotypes and sequevars of R. solanacearum described worldwide. These strains are characterized and maintained by cryoconservation and lyophilization. This collection will constitute a great asset to strengthen and optimize responsiveness in epidemiological surveillance, as molecular tools can be assessed and validated against a great diversity. This work will seek for new diagnostic markers and protocols in order to propose accurate, affordable, and convenient technologies suitable for these regions. Data originating from the population diversity study and the development of microsatellite markers will be strategically used for new application-oriented diagnostics. Furthermore, these tools and protocols will be shared within the SWIO and Eastern African laboratories. 
The genotyping data will be shared among partners and eventually been made public once scientific papers are released. For this, we will use the MLVA bank 8 of 21 database (http://www.biopred.net/MLVA/) which has been developed by two of the partners (UMR PVBMT and RPB/IPME) of the present project during a previous ANR-funded project (ANR-2010-BLANC-1723). The website allows viewing databases with sorting and clustering options, submitting queries, and sharing databases which are maintained and managed by different owners once a common agreement is achieved among partners. This database can handle different types of allelic data (MLVA, MLST, CRISPR…).

Finally, strains representative of the genetic diversity of R. solanacearum will be inoculated to solanaceous plant varieties in the P3 facilities held in CIRAD Reunion laboratories, in Kenya and in Mauritius to investigate the phenotypic diversity (virulence and aggressiveness) of the strains and then to identify genetic resources for resistance to bacterial wilt.