Source identification of brown rot and ring rot infections in Belgium (1989-2021) by forensic investigation of bacterial genome sequences

General introduction

To determine the original source of infection over the past three decades from water samples and batches of potatoes contaminated with brown rot (Ralstonia solanacearum) and ring rot bacteria (Clavibacter sepedonicus), the SourceTrack project used a reverse contact tracing approach with advanced DNA sequencing techniques. Forensic analysis of the bacterial genomes present in the sample collection has revealed genetic diversity in the Ralstonia solanacearum and Clavibacter sepedonicus collections. However, to definitively determine the source isolate, the data still requires a method that measures genetic changes over time (i.e., a molecular clock).

For more than 30 years, both quarantine bacteria have posed a threat to the cultivation of potatoes in particular, but also to tomatoes and eggplants. Absolutely no infected fruits may be found in export consignments. Farmers in a number of regions ('protection zones') are still not allowed to use surface water for crop irrigation due to risk assessments based on annual samples taken from watercourses. ILVO has stored all samples collected between 1989 and 2021 in Belgian watercourses and from the bittercress host plant. The bacteria isolated from contaminated potato batches at the time have also been stored. In this project, the samples were analyzed and compared for the first time using new technology.

Research approach

Genetic analyses were performed on isolates of Ralstonia and Clavibacter obtained over the years and stored in a culture collection. Whole Genome Sequencing (WGS) forms the basis for microbial genotyping in outbreak studies. The genomic data obtained via WGS were then analyzed using various forensic methods to identify small genetic differences. Genetic diversity was measured by (1) gene-by-gene allele determination in the core and accessory genome (cgMLST and agMLST), (2) diversity of the number of repeats of short sequences in specific genomic loci (MLVA), and (3) analysis of the variations of individual nucleotides in the genome (SNP calling).

Relevance/Valorisation

Based on the bacterial genomes, genetic diversity could be demonstrated in the collections. Despite the uneven resolution of the methods and their varying value for the bacterial species studied, similar conclusions were obtained about distribution patterns and epidemiological connections by combining isolates into phylogenomic clusters. However, it was not yet possible to determine the source isolate. It should also be possible to move from the static processing of snapshots from an epidemiology, often months after the findings, to a situation in which epidemiological data are processed in real time. This would allow intervention upon detection, rather than documenting it in retrospect as was done in this study.