Differentiation of Pseudomonas solanacearum, Pseudomonas syzygii, pseudomonas pickettii and the Blood Disease Bacterium by partial 16S rRNA sequencing: construction of oligonucleotide primers for sensitive detection by polymerase chain reaction

Abstract

The sequence of a 292 bp segment of the DNA encoding 16s rRNA (corresponding to positions 44-337 of the Escherichia coli 16s rRNA sequence) was determined for each of 40 Pseudomonas solanacearum, four banana Blood Disease Bacterium, three P. syzygii and two P. pickettii strains. Phylogenetic relationships derived from comparison of these sequences to each other, and to equivalent 16s rRNA gene sequences from other bacteria present in the EMBL databank, conform well with those obtained previously by DNA-DNA/rRNA hybridization experiments. The 16s rRNA sequence of the Blood Disease Bacterium was identical over the 292 bp to one of the four sequence groups of P. solanacearum, suggesting that these pseudomonads are more closely related to each other than to P. syzygii or P. pickettii. Sequence data comparisons allowed construction of an oligonucleotide specific for P. solanacearum, P. syzygii and the Blood Disease Bacterium. Use of the specific oligonucleotide with a non-specific oligonucleotide in the polymerase chain reaction enabled 1-10 cells of bacteria in this group to be detected after 50 rounds of amplification by visualizing a 287-288 bp product on agarose gels.

Epidemiological analysis of clones and cultivars of potato in soil naturally infested with Ralstonia solanacearum biovar 2

“…However, some tubers were positive for R. solanacearum by DAS-ELISA and PCR, indicating the latent nature of the infection in resistant genotypes as well as in susceptible ones (Table 4) Using the same DNA extraction method and primers (Oli 1 and Y2) as were used by us, Llop et al (1999) found that PCR was more efficient at detecting latent infections of potato tubers as compared to ELISA and immunofluorescence tests using polyclonal anti-sera or traditional microbiological methods, with PCR being able to detect as few as 100 colony forming units (CFU).mL aimed at minimizing dissemination of the pathogen through infected tubers (Elphinstone et al, 1996;Priou et al, 1999;Llop et al, 1999;Martins, 2000). Seal et al (1993) reported that PCR can detect as few as one to ten R. solanacearum cells, although this level of sensitivity could not be reproduced by other workers (Elphinstone et al, 1996;Martins, 2000). New methods have produced an increase in the sensitivity of detection techniques, an example being the use of a pretreatment protocol involving semi-selective enrichment medium plus the addition of 0.05 M NaOH and heating at 100 °C for 6 minutes during the PCR (Elphinstone et al, 1996).…”

“…The tests carried out were the double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using Polyclonal anti-serum related to R. solanacearum (Castro et al. 1993) and the polymerase chain reaction (PCR) using the Oli 1 and Y2 primers (Seal et al. 1993).…”

“…Briefly, the polymerase chain reaction (PCR) was performed in a total volume of 25 µL of PCR buffer solution (10 mM Tris-HCl (pH8.3) 50 mM KCl, 3mM of MgCl 2 ), 0.2 mM of each deoxynucleotide (Invitrogen, Brazil), 1.25 units of AmpliTaq polymerase (Gibco-BRL, Brazil), 1 µM of the primers OLI1 (5´GGG GGT AGC TTG CTA CCT GCC3´) and Y2 (5´CCC ACT GCT GCC TCC CGT AGG AGT3´) (Cybersyn, USA) (Seal et al, 1993) and 3 µL of diluted sample DNA. Amplification was performed in a Minicycler™ thermocycler (MJ Research, USA) at 96 °C for 2 min followed by 35 cycles of 94 ºC for 20 s, 68 °C for 20 s and 72 °C for 30 s, with a final 10 min extension at 72 °C.…”

Diversidade genética de isolados de Ralstonia solanacearum e caracterização molecular quanto a filotipos e sequevares

“…A identificação da espécie pode ser facilmente realizada por meio de oligonucleotídeos iniciadores, como o OLI1/Y2, utilizado com sucesso para identificação de isolados de R. solanacearum e espécies relacionadas (R. syzygii e Blood Disease Bacterium) (Seal et al, 1993;Arahal et al, 2004). Já os oligonucleotídeos PS96H/I são específicos para R. solanacearum (Seal et al, 1992); entretanto, o desconhecimento da região-alvo desse iniciador no genoma tem implicado em sua baixa utilização (Arahal et al, 2004).…”

Occurrence and diversity of Ralstonia solanacearum populations in Brazil

“…Several PCR protocols and specific primers were designed for detection or identification of the species and for phylotyping ( Table 2) (SEAL et al, 1993;ELPHINSTONE et al, 1996;OPINA et al, 1997;FEGAN et al, 1998;BOUDAZIN et al, 1999;PASTRIK;MAISS, 2000;LUISETTI, 2000;WELLER et al, 2000). Classification into phylotypes is done by PCR Multiplex with the Nmult series primers (based on ITS region) and the classification into sequevar by partially sequencing gene egl (encoding the enzyme endoglucanase).…”