Evaluation of somatic hybrids of potato with Solanum stenotomum after a long-term in vitro conservation.

Somatic hybrids of potato with a cultivated relative, Solanum stenotomum also called Solanum tuberosum Stenotomum group, were evaluated for their physiological and agronomical characteristics as well as the stability of the introgressed resistance to bacterial wilt, caused by Ralstonia solanacearum, after a long-term in vitro conservation for more than 5 years. Analysis of photosynthesis showed that the PEPC/Rubisco ratio remained lower than 0.5 for all vitroplants of potato and the somatic hybrids, except for the relative species. This indicates that the carbon metabolism is heterotrophic (ratio>1) for S. stenotomum, and autotrophic for potato and the somatic hybrids (ratio<1). In both in vitro and greenhouse conditions, potato and the somatic hybrids produced few bigger tubers, while many small tubers were obtained from the relative. The hybrid tubers were morphologically intermediate. The starch content of hybrid tubers was much lower than that of potato, but similar to that of the relative species. Interestingly, the level of bacterial resistance, introgressed from S. stenotomum into potato, was shown to be very stable and remained as high as that of the relative after a long-term period of in vitro conservation.

Evaluation of procedures for reliable PCR detection of Ralstonia solanacearum in common natural substrates.

Several procedures were compared for reliable PCR detection of Ralstonia solanacearum in common substrates (plant, seed, water and soil). In order to prevent the inhibition of PCR by substances contained in crude extracts, numerous DNA extraction procedures as well as additives to buffers or PCR mixtures were checked. Our results showed that the efficiency of these methods or compounds depended greatly upon the nature of the sample. Consequently, preparation of samples prior to PCR depended upon sample origin. Simple methods such as a combined PVPP/BSA treatment or the association of filtration and centrifugation for detecting the bacterium in plant or water samples were very powerful. DNA capture also efficiently overcame PCR inhibition problems and ensured the detection of R. solanacearum in environmental samples. However, the commercial DNA extraction QIAamp kit appeared to be the most effective tool to guarantee the accurate PCR detection of the pathogen whatever the origin of the sample; this was particularly true for soil samples where the commonly used methods for the detection of R. solanacearum were inefficient. This study demonstrates that using an appropriate procedure, PCR is a useful and powerful tool for detecting low levels of R. solanacearum populations in their natural habitats.

Genetic diversity of Ralstonia solanacearum as assessed by PCR-RFLP of the hrp gene region, AFLP and 16S rRNA sequence analysis, and identification of an African subdivision.

The genetic diversity among strains in a worldwide collection of Ralstonia solanacearum, causal agent of bacterial wilt, was assessed by using three different molecular methods. PCR-RFLP analysis of the hrp gene region was extended from previous studies to include additional strains and showed that five amplicons were produced not only with all R. solanacearum strains but also with strains of the closely related bacteria Pseudomonas syzygii and the blood disease bacterium (BDB). However, the three bacterial taxa could be discriminated by specific restriction profiles. The PCR-RFLP clustering, which agreed with the biovar classification and the geographical origin of strains, was confirmed by AFLP. Moreover, AFLP permitted very fine discrimination between different isolates and was able to differentiate strains that were not distinguishable by PCR-RFLP. AFLP and PCR-RFLP analyses confirmed the results of previous investigations which split the species into two divisions, but revealed a further subdivision. This observation was further supported by 16S rRNA sequence data, which grouped biovar 1 strains originating from the southern part of Africa.