Application of Variable-Number Tandem-Repeat Typing To Discriminate Ralstonia solanacearum Strains Associated with English Watercourses and Disease Outbreaks

Abstract

b Variable-number tandem-repeat (VNTR) analysis was used for high-resolution discrimination among Ralstonia solanacearum phylotype IIB sequevar 1 (PIIB-1) isolates and further evaluated for use in source tracing. Five tandem-repeat-containing loci (comprising six tandem repeats) discriminated 17 different VNTR profiles among 75 isolates from potato, geranium, bittersweet (Solanum dulcamara), tomato, and the environment. R. solanacearum isolates from crops at three unrelated outbreak sites where river water had been used for irrigation had distinct VNTR profiles that were shared with PIIB-1 isolates from infected bittersweet growing upriver of each site. The VNTR profiling results supported the implication that the source of R. solanacearum at each outbreak was contaminated river water. Analysis of 51 isolates from bittersweet growing in river water at different locations provided a means to evaluate the technique for studying the epidemiology of the pathogen in the environment. Ten different VNTR profiles were identified among bittersweet PIIB-1 isolates from the River Thames. Repeated findings of contiguous river stretches that produced isolates that shared single VNTR profiles supported the hypothesis that the pathogen had disseminated from infected bittersweet plants located upriver. VNTR profiles shared between bittersweet isolates from two widely separated Thames tributaries (River Ray and River Colne) suggested they were independently contaminated with the same clonal type. Some bittersweet isolates had VNTR profiles that were shared with potato isolates collected outside the United Kingdom. It was concluded that VNTR profiling could contribute to further understanding of R. solanacearum epidemiology and assist in control of future disease outbreaks.

New Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Scheme for Fine-Scale Monitoring and Microevolution-Related Study of Ralstonia pseudosolanacearum Phylotype I Populations

“…within the Ivory Coast and Africa (41), addressed the global surveillance of phylotype III (52), and identified sources of contamination of the Thames by phylotype IIB-1 (17). These studies improved the comprehension of RSSC epidemiology, its spread, its structuration on different scales, and its colonization pattern.…”

“…The proposed new MLVA scheme is the fourth one developed for the RSSC, after the initial work of N=Guessan et al (including the phylotype I-specific MLVA-13 scheme) (20), the phylotype IIB-1-specific scheme of Parkinson et al (17), and the phylotype III-specific scheme of Ravelomanantsoa et al (52). These three schemes, used on regional scales, allowed the inference of possible dissemination routes of phylotype I and Vallon 2012 (n ϭ 319) collections were genotyped using the MLVA-7 scheme (left), and their distribution within the three collections was determined using the table.value tool (right).…”

“…Unfortunately, the low repeatability and reproducibility with RAPD markers make the results difficult to interpret, and REP-PCR portability between laboratories remains problematic (6). To date, three MLVA schemes have already been developed, to decipher RSSC population genetics on different scales (41), to investigate sources of water contamination by Ralstonia solanacearum (phylotype IIB) (17), and to characterize African R. pseudosolanacearum phylotype III strains on local and global scales (52). The MLVA phylotype I-specific scheme of N=Guessan et al (20) (13 loci; MLVA-13) and the MLVA phylotype III-specific scheme of Ravelomanantsoa et al (52) gave promising results in discriminating populations on scales ranging from cropping areas to continents (41).…”

“…Although there is a growing interest in the use of MLVA on plant-associated bacteria, only a few studies have been published, focusing on their population biology. These studies aimed at tracing sources of water contamination (Ralstonia solanacearum [17]), assessing the host-driven pathogen population structure (Pseudomonas syringae pv. maculicola and P. syringae pv.…”

Discrimination of Ralstonia solanacearum isolates by genetic signatures produced by single-strand conformation polymorphism and low-stringency single specific primer PCR analysis

“…Diversity within the R. solanacearum isolates has been studied from decades using techniques such as 16S rRNA, AFLP, RFLP, BOX-PCR, rep-PCR and multiplex PCR (Wicker et al, 2007;Parkinson et al, 2013). Even though these techniques are specific, sensitive and rapid but not free from difficulties such as additional enzymes, instrumentation and high cost, are difficult to adopt in developing countries when screening large number of isolates.…”