The broad-host-range plant pathogen Ralstonia solanacearum
causes serious crop losses around the world. Potato (Solanum tuberosum
) is especially vulnerable to the pathogen, which induces severe wilt and tuber rot symptoms. One particular subgroup of R. solanacearum
(historically known as race 3 biovar 2) is a high-concern quarantine pest, the accidental introduction of which has serious economic and political consequences. The pathogen is therefore the subject of statutory control measures in the European Union, the United States, and many other countries worldwide, which aim to monitor and prevent its spread and direct eradication measures where needed. The characteristics of the pathogen and its hosts and pathology have been extensively reviewed (1–3
). Aspects of race 3 biovar 2 biology that concern cold tolerance relative to other taxa in the species have also been studied, and these factors may be important in predicting the geographical range of the pathogen (4
). Phylogenetic analysis of the species, based on comparison of partial endoglucanase (egl
) and other gene sequences, found considerable intraspecies diversity, and four major phylogroups with distinct geographical centers of origin have been identified (6–8
). Phylogroup II strains were found to have a center of origin in South America. R. solanacearum
race 3 biovar 2 isolates are represented by a single egl
sequevar within phylogroup IIB, designated PIIB sequevar 1 (abbreviated as PIIB-1 here) (8
). This strain has been disseminated around the world (6
) in infected potatoes and has been isolated on potatoes in several European countries over the last 2 decades (3
). In Europe, R. solanacearum
isolates from field crops of potato and tomato (Solanum lycopersicum
) have all been identified as PIIB-1, and in this region, non-PIIB-1 strains are rare and restricted to various greenhouse crops (10
). The same PIIB-1 strain was also introduced into the United States and Europe on infected geranium (Pelargonium
) cuttings (11
Diversity within the taxon has been widely studied using a range of other analyses, such as whole-cell fatty acid profiling (13
), metabolic profiling (14
), 16S rRNA sequencing (15
), and various genetic fingerprinting methods, including amplified fragment length polymorphism (AFLP) (16
), rare-cutting pulsed-field gel electrophoresis (RC-PFGE) (18
), repetitive sequence-based PCR (rep-PCR) (17
), restriction fragment length polymorphisms of pathogenicity genes (16
), and multilocus sequence typing (20
). All the studies concluded that there is little or no diversity within PIIB-1 isolates from worldwide sources, and the taxon has been described as nearly clonal. This conclusion is supported by the extremely high level of homology between independently sequenced genomes of two PIIB-1 isolates (21
). The lack of discrimination among PIIB-1 isolates collected from a wide geographic distribution has been attributed to their relatively recent international dissemination through centralized trade of vegetatively propagated potatoes infected with the strain.
To date, there have been seven confirmed outbreaks of brown rot disease caused by R. solanacearum
PIIB-1 in potato crops in England; two in different locations in the Thames Valley (in 1992 and 1995), two in Northamptonshire in 1999, one in Kent in 2000, one in Nottinghamshire in 2005, and one in 2009 where an infected imported seed stock was planted in Somerset. The PIIB-1 strain has also caused two outbreaks of tomato bacterial wilt in heated greenhouse crops grown at one locality in Bedfordshire (in 1997 and 1998). The tomato crops in which there were outbreaks were also directly irrigated from a watercourse contaminated with R. solanacearum
. The pathogen is known to persist in rivers and other watercourses by infecting riparian woody nightshade, or bittersweet (Solanum dulcamara
), with aquatic roots growing out from the river banks (9
). The plant is common along the banks of the Thames and other rivers in southern England, and mature plants of the perennial can form a dense tangle of semisubmerged stolons. Infection of bittersweet is therefore considered to be an important means of establishment and persistence of the PIIB-1 strain following introduction into ecosystems in northern Europe (5
The high mutation rate associated with tandemly repeated DNA sequences has been exploited in producing highly discriminatory variable-number tandem-repeat (VNTR) profiles, which are used extensively in the forensic identification of individuals and discrimination among close relatives (26
). Prokaryote VNTR structure and diversity has been reviewed (27
). Analysis of sequenced bacterial genomes has found that tandem-repeat sequences are frequent and widespread. VNTR analysis has resolved very closely related strains to facilitate many epidemiological studies of human and animal diseases, including anthrax, tuberculosis, and salmonellosis (29
). Studies using VNTR-based analyses to differentiate among strains of phytopathogenic bacteria and to provide epidemiological information have not been extensively reported. Two studies on the occurrence of VNTR-containing loci in Xylella fastidiosa
) and Pseudomonas syringae
) have been reported. At the time of writing, a comparative analysis of the occurrence of VNTR-containing loci within the phylotypes of R. solanacearum
, including PIIB-1 strains, had become available (34
There were three main objectives of this first application of VNTR to study R. solanacearum PIIB-1 associated with disease outbreaks. The first was to produce VNTR profiles from a panel of 12 PIIB-1 reference isolates from diverse sources and to evaluate them as a means to produce a highly discriminatory strain identification scheme among these closely related isolates. The second objective was to establish the utility of VNTR profiling in source tracing. To accomplish this, VNTR profiles for isolates from infected crops and for environmental isolates collected upriver on watercourses used to irrigate the crops, which were implicated as sources of the pathogen, were compared. The third objective was to ascertain whether VNTR profile analysis would provide additional epidemiological information on the pathogen's colonization, survival, and spread along the River Thames catchment. This study involved comparing VNTR profiles of isolates collected from bittersweet growing along the River Thames following the first known brown rot outbreaks in potato during 1992 and 1996. To investigate the longevity of infection over time in bittersweet, the site of earlier isolations was revisited and new strains were isolated. The VNTR profiles of the newly isolated strains were then compared with those isolated 15 years previously from the same stretch of river.
The first objective of this study was to establish whether the technique could be used to discriminate different VNTR profile types among reference isolates of R. solanacearum
PIIB-1. The 17 distinct VNTR profiles that were identified clearly provide an efficient means for discriminating between PIIB-1 strains. Considering the recognized lack of diversity among PIIB-1 strains (13–20
), the technique provides a very high level of strain discrimination.
This VNTR analysis presents a valuable opportunity to identify individual populations within the PIIB-1 group of R. solanacearum
. This VNTR study of PIIB-1 strains used a limited set of loci, and future studies using additional loci may extend the discriminatory power of the technique still further. Recently, a VNTR scheme for discriminating Listeria monocytogenes
has been optimized by combining tandem-repeat-containing loci from four previously reported schemes (37
). At the time of writing, a study had been reported (34
) that provides an analysis of VNTR loci throughout R. solanacearum
. In agreement with our study, this study also found that VNTR profiles could discriminate PIIB-1 strains. Four discriminative loci identified 10 VNTR profile types among 31 PIIB-1 strains analyzed. Additionally, the study found VNTR loci were strongly associated with phylogroups and that PIIB-1 loci were rarely present in genomes of the other phylotypes. The availability of the genome sequence (21
) from the representative PIIB-1 strain (UW551) was an essential step in identifying the discriminatory VNTR loci in our study. This confirms the importance of identifying VNTR-containing loci using a genome sequence derived from a strain very closely related to those under epidemiological investigation.
The second aim of this study was to evaluate the use of VNTR profiling in source tracing. This was done by investigating three isolated disease outbreaks for which R. solanacearum
isolates were available from affected crops, as well as associated river water and bittersweet previously implicated as sources of the pathogen. This provided a functional evaluation of the suitability of VNTR profiling in source tracing. For successful source tracing, the VNTR scheme should be sufficiently discriminatory to identify clonal types, but at the same time, the VNTR profiles should be sufficiently stable to provide consistent profiles within an outbreak. VNTR profiles obtained from strains isolated at the three outbreaks were specific to each outbreak. In each case, the profiles of isolates from bittersweet growing upriver matched those from the affected crops, thus supporting the potential utility of VNTR profiling in source tracing. This is consistent with the utility of VNTR analyses in epidemiological studies with human pathogens (29
). VNTR profile stability within an outbreak of Salmonella enterica
was studied using 190 isolates, and only small changes in profiles were found during the course of the outbreak (37
). Similarly, the optimized L. monocytogenes
VNTR scheme (38
) used to analyze strains from an outbreak found one of the nine loci was variable. In our study, two similar profile types (P11 and P12) were associated with the outbreak on farm 1, both of which were found in bittersweet at ditch 1 on the farm and immediately upstream from farm 1 in the Thames. Source-tracing studies should therefore examine multiple isolates to gain a clear picture of the diversity of types involved in an outbreak.
The third objective of the study was to determine whether VNTR profiling could provide epidemiological information on PIIB-1 colonization of bittersweet in the Thames Valley to further previous studies on the pathogen's survival and spread (39
). All four bittersweet isolates from stretch S3 sampled during 2008 were identified as P13, which was the predominant profile in this stretch of the Thames when they were isolated during 1993 and 1994 some 14 or 15 years earlier. This suggests long-term associations of strains with specific VNTR profiles with bittersweet during this period and also that VNTR profiles may be stable over many years in the Thames environment. The shared (P8) profile common to bittersweet strains isolated from stream 1 in 1993 and from the same host from Sweden (1975) approximately 18 years earlier also points to long-term stability of the profile. However, the possibility remains that strains with common profiles could be derived independently by mutation from strains with different profiles and origins.
It is also possible that some VNTR profile diversification has occurred within the Thames Valley. Three VNTR profiles (P11, P12, and P13) were isolated from contiguous stretches of the Thames (stretches S3 and S5), and each contained the same 4- and 6-repeat combination for the double-tandem-repeat-containing locus L578. These profiles, which have so far been found only in this region of the Thames, may have diversified here, which would explain why two profile types were associated with the outbreak at farm 1. However, the diversity of VNTR profiles identified along the whole stretch of the Thames suggests multiple contamination events have probably occurred. The possibility of multiple contamination events in the River Thames catchment is consistent with the theory that PIIB-1 strains could have been introduced through discharge into the drainage system from contaminated potato waste originating from domestic use (9
). Shared VNTR profiles among R. solanacearum
isolates (e.g., P2 and P3) from the Thames Valley and from potatoes originating from other countries also support this possible means of introduction.
In contrast to the heterogeneity of VNTR profile types found in R. solanacearum isolates from the Thames, colonization of bittersweet along the River Colne and Ray tributaries upriver from their confluence with the River Thames exclusively comprised a single (P3) VNTR type. Considering the long distance separating the two tributaries, it is possible that independent contamination of the tributaries occurred from a common infection source, e.g., by discharge of the same VNTR profile strain from the same source of contaminated potatoes through independent drainage systems.
The two examples of contiguous river stretches contaminated with strains of only a single VNTR profile (Rivers Colne and Ray) suggests sequential infections of bittersweet from infected plants upriver. Long-term infections of bittersweet and transmission of the pathogen to bittersweet hosts located downriver are factors that are likely to have contributed to this host serving as an intractable infection reservoir.
The lack of diversity within the PIIB-1 group at the sequevar level indicates that these strains have only recently been disseminated around the world and that insufficient time has elapsed for strains to diversify. Intensification and centralization of potato production, together with transport of infected potatoes across international boundaries, would have contributed to the dissemination of PIIB-1 strains. This study provides evidence for the potential value of VNTR profile analysis for source tracing of PIIB-1 outbreaks and for epidemiological analysis of the pathogen in the environment. New information has been gained on the possible pathways of introduction and transmission of PIIB-1 strains from the environment to crops. The application of the technique could assist in the elimination of pathways of introduction of this quarantine pathogen and in the management of future R. solanacearum eradication programs.