In an attempt to isolate the superoxide dismutase (SOD) gene from the anaerobic sulfate-reducing bacterium Desulfoarculus baarsii, a DNA fragment was isolated which functionally complemented an Escherichia coli mutant (sodA sodB) deficient in cytoplasmic SODs. This region carries two open reading frames with sequences which are very similar to that of the rbo-rub operon from Desulfovibrio vulgaris. Independent expression of the rbo and rub genes from ptac showed that expression of rbo was responsible for the observed phenotype. rbo overexpression suppressed all deleterious effects of SOD deficiency in E. coli, including inactivation by superoxide of enzymes containing 4Fe-4S clusters and DNA damage produced via the superoxide-enhanced Fenton reaction. Thus, rbo restored to the sodA sodB mutant the ability to grow on minimal medium without the addition of branched amino acids, and growth on gluconate and succinate carbon sources was no longer impaired. The spontaneous mutation rate, which is elevated in SOD-deficient mutants, returned to the wild-type level in the presence of Rbo, which also restored aerobic viability of sodA sodB recA mutants. Rbo from Desulfovibrio vulgaris, but not Desulfovibrio gigas desulforedoxin, which corresponds to the NH 2 -terminal domain of Rbo, complemented sod mutants. The physiological role of Rbo in sulfate-reducing bacteria is unknown. In E. coli, Rbo may permit the bacterium to avoid superoxide stress by maintaining functional (reduced) superoxide sensitive 4Fe-4S clusters. It would thereby restore enzyme activities and prevent the release of iron that occurs after cluster degradation and presumably leads to DNA damage.
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...
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.
Three cos-type virulent Streptococcus thermophilus phages were isolated from failed mozzarella production in Uruguay. Genome analyses showed that these phages are similar to those isolated elsewhere around the world. The CRISPR1 and CRISPR3 arrays of the three S. thermophilus host strains from Uruguay were also characterized and similarities were noted with previously described model strains SMQ-301, LMD-9 and DGCC7710. Spontaneous bacteriophage-insensitive S. thermophilus mutants (BIMs) were obtained after challenging the phage-sensitive wild-type strain Uy02 with the phage 128 and their CRISPR content was analyzed. Analysis of 23 BIMs indicated that all of them had acquired at least one new spacer in their CRISPR1 array. While 14 BIMs had acquired spacer at the 5′-end of the array, 9 other BIMs acquired a spacer within the array. Comparison of the leader sequence in strains Uy02 and DGCC7710 showed a nucleotide deletion at position -1 in Uy02, which may be responsible for the observed ectopic spacer acquisition. Analysis of the spacer sequences upstream the newly acquired ectopic spacer indicated presence of a conserved adenine residue at position -2. This study indicates that natural strains of S. thermophilus can also acquire spacers within a CRISPR array.
Blue mold caused by Penicillium spp. is the most important postharvest disease of apple in Uruguay. Fourteen isolates of Penicillium were recovered from rotten apple and pear fruit with blue mold symptoms, and from water from flotation tanks in commercial apple juice facilities. Phenotypic identification to species level was performed, and the isolates were tested for sensitivity to commonly used postharvest fungicides. Genetic characterization of the isolates was performed with restriction fragment length polymorphism of the region including the internal transcribed spacer (ITS) ITS1 and ITS2 and the 5.8SrRNA gene (ITS1-5.8SrRNA gene-ITS2) ribosomal DNA region and with random amplified polymorphic DNA (RAPD) primers. Both techniques were able to differentiate these isolates at the species level. RAPD analysis proved to be an objective, rapid, and reliable tool to identify Penicillium spp. involved in blue mold of apple. In all, 11 isolates were identified as Penicillium expansum and 3 as P. solitum. This is the first report of P. solitum as an apple pathogen in Uruguay.
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