Several breeding programs are under way to introduce resistance to bacterial wilt caused by Ralstonia solanacearum in solanaceous crops. The lack of screening methods allowing easy measurement of pathogen colonization and the inability to detect latent (i.e., symptomless) infections are major limitations when evaluating resistance to this disease in plant germplasm. We describe a new method to study the interaction between R. solanacearum and potato germplasm that overcomes these restrictions. The R. solanacearum UY031 was genetically modified to constitutively generate light from a synthetic luxCDABE operon stably inserted in its chromosome. Colonization of this reporter strain on different potato accessions was followed using life imaging. Bacterial detection in planta by this nondisruptive system correlated with the development of wilting symptoms. In addition, we demonstrated that quantitative detection of the recombinant strain using a luminometer can identify latent infections on symptomless potato plants. We have developed a novel, unsophisticated, and accurate method for high-throughput evaluation of pathogen colonization in plant populations. We applied this method to compare the behavior of potato accessions with contrasting resistance to R. solanacearum. This new system will be especially useful to detect latency in symptomless parental lines before their inclusion in long-term breeding programs for disease resistance.Bacterial wilt caused by Ralstonia solanacearum is one of the world's most devastating bacterial diseases of plants (Peeters et al. 2013). It is present worldwide, threatening food safety of small producers in tropical and subtropical areas, especially in China, Bangladesh, Bolivia, and Uganda (Martin and French 1985;Muthoni et al. 2012). However, because the pathogen disseminates easily to long distances via infected plant material (Hayward and Pegg 2013;Janse et al. 2004), there has been a recent spread of the disease to temperate regions (Genin and Boucher 2004). R. solanacearum exhibits an extremely wide host range, causing bacterial wilt on more than 200 plant species, including commercially important crops such as potato, tomato, and banana (Hayward 1991). Wilting symptoms are caused by extensive multiplication of the bacterium in the xylem vessels, which hinders water flow in the plant. In addition, the bacterium can colonize asymptomatically several weeds that act as pathogen reservoirs (Genin and Denny 2012) and survive in waterways and soil for long periods (Caruso et al. 2005;Elphinstone 2005). These facts, together with the lack of resistant commercial varieties in any of its hosts , render the control of bacterial wilt very challenging.R. solanacearum is considered a species complex, divided into four phylotypes which roughly reflect their geographic origin: phylotype I groups strains from Asia, phylotype II from the Americas, phylotype III from Africa, and phylotype IV from Indonesia (Peeters et al. 2013). Each phylotype is subdivided into different sequevars which are clus...

Bacterial wilt (BW) caused by Ralstonia solanacearum is responsible for substantial losses in cultivated potato (Solanum tuberosum) crops worldwide. Resistance genes have been identified in wild species; however, introduction of these through classical breeding has achieved only partial resistance, which has been linked to poor agronomic performance. The Arabidopsis thaliana (At) pattern recognition receptor elongation factor-Tu (EF-Tu) receptor (EFR) recognizes the bacterial pathogen-associated molecular pattern EF-Tu (and its derived peptide elf18) to confer anti-bacterial immunity. Previous work has shown that transfer of AtEFR into tomato confers increased resistance to R. solanacearum. Here, we evaluated whether the transgenic expression of AtEFR would similarly increase BW resistance in a commercial potato line (INIA Iporá), as well as in a breeding potato line (09509.6) in which quantitative resistance has been introgressed from the wild potato relative Solanum commersonii. Resistance to R. solanacearum was evaluated by damaged root inoculation under controlled conditions. Both INIA Iporá and 09509.6 potato lines expressing AtEFR showed greater resistance to R. solanacearum, with no detectable bacteria in tubers evaluated by multiplex-PCR and plate counting. Notably, AtEFR expression and the introgression of quantitative resistance from S. commersonii had a significant additive effect in 09509.6-AtEFR lines. These results show that the combination of heterologous expression of AtEFR with quantitative resistance introgressed from wild relatives is a promising strategy to develop BW resistance in potato.

Ralstonia solanacearum is the agent of bacterial wilt infecting >200 different plant species covering >50 botanical families. The genus R. solanacearum can be classified into four phylotypes and each phylotype can be further subdivided into sequevars. The potato brown rot strains of R. solanacearum from phylotype IIB, sequevar 1 (IIB1), historically known as race 3, biovar 2 strains, are responsible for important economic losses to the potato industry and threaten ornamental crop production worldwide. Sensitive and specific detection methods are required to control this pathogen. This article provides a list of 70 genes and 15 intergenes specific to the potato brown rot strains of R. solanacearum from phylotype IIB1. This list was identified by comparative genomic hybridization on microarray and subsequent polymerase chain reaction validation with 14 IIB1 strains against 45 non-IIB1 strains that covered the known genetic diversity in R. solanacearum. The microarray used consisted of the previously described microarray representative of the phylotype I strain GMI1000, to which were added 660 70-mer oligonucleotides representative of new genomic islands detected in the phylotype IIB1 strain IPO1609. The brown rot strain-specific genes thus identified were organized in nine clusters covering 2 to 29 genes within the IPO1609 genome and 6 genes isolated along the genome. Of these specific genes, 29 were parts of mobile genetic elements. Considering the known instability of the R. solanacearum genome, we believe that multiple probes are required to consistently detect all IIB1 strains and we recommend the use of probes which are not part of genetic mobile elements.

Bacterial wilt, caused by Ralstonia solanacearum, is a major disease affecting potato (Solanum tuberosum) production worldwide. Although local reports suggest that the disease is widespread in Uruguay, characterization of prevalent R. solanacearum strains in that country has not been done. In all, 28 strains of R. solanacearum isolated from major potato-growing areas in Uruguay were evaluated, including 26 strains isolated from potato tubers and 2 from soil samples. All strains belonged to phylotype IIB, sequevar 1 (race 3, biovar 2). Genetic diversity of strains was assessed by repetitive-sequence polymerase chain reaction, which showed that the Uruguayan strains constituted a homogeneous group. In contrast, inoculation of the strains on tomato and potato plants showed, for the first time, different levels of aggressiveness among R. solanacearum strains belonging to phylotype IIB, sequevar 1. Aggressiveness assays were also performed on accessions of S. commersonii, a wild species native to Uruguay that is a source of resistance for potato breeding. No significant interactions were found between bacterial strains and potato and S. commersonii genotypes, and differences in aggressiveness among R. solanacearum strains were consistent with previously identified groups based on tomato and potato inoculations. Moreover, variation in responses to R. solanacearum was observed among the S. commersonii accessions tested.

Solanum commersonii is a wild tuber-bearing species native to Uruguay with high potential for use in potato breeding programs. Little is known about the genetic diversity within this wild species and the relationship with the resistance to the bacterial pathogen Ralstonia solanacearum. We studied 30 S. commersonii clonal accessions, 20 of which were collected from geographically diVerent areas across the country, while the other ten were grown from seeds from a single plant. Resistance against R. solanacearum was tested and diVerent levels of resistance were found, ranging from delayed wilting to asymptomatic reactions. The genetic variation and the relationships among individuals in this germplasm collection were studied by diVerent molecular markers: Random AmpliWed Polymorphic DNA (RAPD), AmpliWed Fragment Length Polymorphism (AFLP) and Microsatellites or Simple Sequence Repeats (SSR). AFLP markers generated the largest number of total and polymorphic fragments per assay unit while SSR revealed the highest frequency of polymorphic bands (100%), followed by AFLP (96.2%) and RAPD (89.4%). In contrast, when comparing the number of diVerent genetic proWles generated, the SSR markers exhibited the lowest discriminatory power. The clustering pattern obtained with the three marker systems showed a similar distribution of the S. commersonii germplasm revealing a high correlation between the three methods employed. All three dendrograms grouped most of the accessions into two main clusters, containing the same accessions regardless of the marker type. Bacterial wilt resistant accessions were present in both clusters. Accessions originated from diVerent seeds of the same plant were grouped within one of the major clusters, and diVered in the response to R. solanacearum revealing segregation of resistance. Furthermore, the distribution in two main clusters showed high correspondence with the geographical origin of the accessions, from the north and south of the country, and with the subspecies malmeanum and commersonii morphologically identiWed.