J.M. van der Wolf

Fluobodies : green fluorescent single-chain Fv fusion proteins

An expression system (pSKGFP), which permits the expression of single-chain variable fragments as fusion proteins with modified green fluorescent proteins, was designed. This expression system is comparable to frequently used phage display vectors and allows single-step characterization of the selected recombinant antibodies by flow cytometry or fluorescent cell staining. Two different single-chain variable fragment antibodies, both directed against the lipopolysaccharide of the bacterium Ralstonia solanacearum have been genetically fused to a red-shifted green fluorescent protein and the produced fusion protein tested for usefulness. These fluobodies can be produced in cultures of bacterial cells and purified using immobilized metal affinity chromatography. They function well in flow cytometry and immunofluorescent cell staining, are specific for their target antigens and, unlike FITC-conjugated antibodies, they do not fade upon illumination.

Systemic colonization of potato plants by a soilborne, green fluorescent protein-tagged strain of Dickeya sp. biovar 3.

ABSTRACT Colonization of potato plants by soilborne, green fluorescent protein (GFP)-tagged Dickeya sp. IPO2254 was investigated by selective plating, epifluorescence stereo microscopy (ESM), and confocal laser scanning microscopy (CLSM). Replicated experiments were carried out in a greenhouse using plants with an intact root system and plants from which ca. 30% of the lateral roots was removed. One day after soil inoculation, adherence of the pathogen on the roots and the internal colonization of the plants were detected using ESM and CLSM of plant parts embedded in an agar medium. Fifteen days post-soil inoculation, Dickeya sp. was found on average inside 42% of the roots, 13% of the stems, and 13% of the stolons in plants with undamaged roots. At the same time-point, in plants with damaged roots, Dickeya sp. was found inside 50% of the roots, 25% of the stems, and 25% of the stolons. Thirty days postinoculation, some plants showed true blackleg symptoms. In roots, Dickeya sp. was detected in parenchyma cells of the cortex, both inter- and intracellularly. In stems, bacteria were found in xylem vessels and in protoxylem cells. Microscopical observations were confirmed by dilution spread-plating the plant extracts onto agar medium directly after harvest. The implications of infection from soilborne inoculum are discussed.

Detection of Clavibacter michiganensis subsp. sepedonicus by AmpliDet RNA, a new technology based on real time monitoring of NASBA amplicons with a molecular beacon

Aims: To develop a procedure for direct detection of viable cells of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, based on AmpliDet RNATM, in which amplicons generated by nucleic acid sequence based amplification (NASBA) are monitored in real time with a molecular beacon.

Amplification of RNA by NASBA allows direct detection of viable cells of Ralstonia solanacearum in potato

Aims: The objective of this study was to develop a Nucleic Acid Sequence Based Amplification (NASBA) assay, targeting 16S rRNA sequences, for direct detection of viable cells of Ralstonia solanacearum, the causal organism of bacterial wilt. The presence of intact 16S rRNA is considered to be a useful indicator for viability, as a rapid degradation of this target molecule is found upon cell death. 
Methods and Results: It was demonstrated by RNase treatment of extracted nucleic acids from R. solanacearum cell suspensions that NASBA exclusively detected RNA and not DNA. The ability of NASBA to assess viability was demonstrated in two sets of experiments. In the first experiment, viable and chlorine‐killed cells of R. solanacearum were added to a potato tuber extract and tested in NASBA and PCR. In NASBA, only extracts spiked with viable cells resulted in a specific signal after Northern blot analysis, whereas in PCR, targeting 16S rDNA sequences, both extracts with viable and killed cells resulted in specific signals. In the second experiment, the survival of R. solanacearum on metal strips was studied using NASBA, PCR‐amplification and dilution plating on the semiselective medium SMSA. A positive correlation was found between NASBA and dilution plating detecting culturable cells, whereas PCR‐amplification resulted in positive reactions also long after cells were dead. 
The detection level of NASBA for R. solanacearum added to potato tuber extracts was determined at 104 cfu per ml of extract, equivalent to 100 cfu per reaction. With purified RNA a detection level of 104 rRNA molecules was found. This corresponds with less than one bacterial cell, assuming that a metabolically active cell contains ca 105 copies of rRNA. Preliminary experiments demonstrated the potential of NASBA to detect R. solanacearum in naturally infected potato tuber extracts. 
Conclusions: NASBA specifically amplifies RNA from viable cells of R. solanacearum even present in complex substrates at a level of 100 cfu per reaction. 
Significance and Impact of the Study: The novel NASBA assay will be particularly valuable for detection of R. solanacearum in ecological studies in which specifically viable cells should be determined.

An internal control for the diagnosis of Crown Gall by PCR

The addition of an internal control (IC) at the appropriate concentration enables recognize of false negatives in the detection of Agrobacterium tumefaciens and improves the reliability of PCR for crown gall diagnosis. Co-amplification of the IC and target sequence from A. tumefaciens ensures the attainment of at least one amplification product in every PCR reaction if the DNA extracted is of high enough quality to be amplified.

Evaluation of phenotypic and molecular typing techniques for determining diversity in Erwinia carotovora subsp. atroseptica

A number of phenotypic and molecular fingerprinting techniques, including physiological profiling (Biolog), restriction fragment length polymorphism (RFLP), enterobacterial repetitive intergenic consensus (ERIC) and a phage typing system, were evaluated for their ability to differentiate between 60 strains of Erwinia carotovora ssp. atroseptica (Eca) from eight west European countries. These techniques were compared with other fingerprinting techniques, random amplified polymorphic DNA (RAPD) and Ouchterlony double diffusion (ODD), previously used to type this pathogen. Where possible, data were represented as dendrograms and groups/subgroups of strains identified. Simpsons index of diversity (Simpsons D) was used to compare groupings obtained with the different techniques which, with the exception of Biolog, gave values of 0·46 (RFLP), 0·39 (ERIC), 0·83 (phage typng), 0·82 (RAPD) and 0·26 (ODD). Of the techniques tested, phage typing showed the highest level of diversity within Eca, and this technique will now form the basis of studies into the epidemiology of blackleg disease.

Genetic diversity of Ralstonia solanacearum race 3 in Western Europe determined by AFLP, RC-PFGE and Rep-PCR

Recently, several outbreaks of brown rot caused by Ralstonia solanacearum (Smith) Smith biovar 2, race 3 were reported in Western Europe. Knowledge of the variation in the population of the target pathogen forms the basis of epidemiological studies and control measures. In this study, the genetic diversity of race 3 strains from France, The Netherlands and the UK was determined with different molecular techniques. AFLP and pulsed-field gel electrophoresis of genomic DNA digested by rare-cutting restriction enzymes (RC-PFGE) were able to separate race 3 strains in different groups, indicating that several clonal lines are present in Europe. ERIC- and BOX-PCR were less discriminative. No correlation was found between geographical origin and profiles obtained.

Immunofluorescence Colony-staining (IFC) for Detection and Quantification of Ralstonia (Pseudomonas) solanacearum Biovar 2 (Race 3) in Soil and Verification of Positive Results by PCR and Dilution Plating

A procedure was developed for specific and sensitive quantitative detection of Ralstonia (Pseudomonas) solanacearum biovar 2 (race 3) in soil. It is based on immunofluorescence colony-staining (IFC) followed by confirmation of the identity of fluorescent colonies by PCR-amplification or dilution plating on a semi-selective medium, SMSA. Addition of sucrose and the antibiotics cycloheximide and crystal violet to the non-selective trypticase soy broth agar resulted in increased colony size and staining intensity of R. solanacearum in IFC. Verification of IFC-results by picking cells from IFC-positive colonies followed by dilution plating of the suspended cells on SMSA was highly efficient. The success rate was 92% and 96% with ‘spiked’ and naturally contaminated soils respectively. Several other bacterial species which cross-reacted with polyclonal antibodies in IFC also grew on SMSA and were difficult to distinguish from R. solanacearum, thereby necessitating confirmation of the results. Rapid verification of IFC-positive results directly by PCR-amplification with primers D2/B specific to division 2 of R. solanacearum had a success rate of 86% and 96% with ‘spiked’ and naturally contaminated soil samples, respectively. Primers D2/B reacted with all R. solanacearum division 2 strains, and strains of R. syzygii and the banana blood disease bacterium, but not with saprophytic bacteria cross-reacting in IFC with R. solanacearum antibodies. In comparative tests, IFC was able to detect consistently ca. 100 cfu g−1 of soil, a detection level similar to that found with direct plating on SMSA, but less laboriously, whereas detection level with a bioassay on tomato plants was only 104−105 cfu g−1 of soil.

Serological characterization of fluorescent Pseudomonas strains cross-reacting with antibodies against Erwinia chrysanthemi

Sixteen bacterial strains, cross-reacting with antibodies againstErwinia chrysanthemi (Ech), were isolated from potato peel extracts, ditch water, and the rhizosphere of wheat, onion, sugar beet and chicory using the immunofluorescence colony-staining procedure. Based on fatty acid profiles, isolates were classified as belonging to thePseudomonas fluorescens group.These strains, together with two previously isolated cross-reactingP. fluorescens strains, crossreacted with polyclonal antibodies against Ech in immunofluorescence cell-staining, Ouchterlony double diffusion, and ELISA. Seventeen strains also reacted strongly with monoclonal antibodies against the lipopolysaccharides (LPS) of Ech in ELISA.Cell envelopes (CE) and proteinase-K-treated CE (mainly LPS) of cross-reacting bacteria were further characterized with SDS-PAGE and Western blotting. Based on CE protein and LPS patterns, the cross-reacting bacteria were classified into two groups, each existing of two subgroups. Both CE and proteinase-K-resistant antigens strongly cross-reacted on immunoblots with antisera against a wild type strain of Ech. With an antiserum against a LPS O-chain lacking mutant of Ech only protein bands but no proteinase-K-resistant antigens were detected on immunoblots. These data suggest that in all cases the highly antigenic LPS O-chain is responsible for the cross-reactions.

Downward vascular translocation of a green fluorescent protein-tagged strain of Dickeya sp. (Biovar 3) from stem and leaf inoculation sites on potato.

Translocation of a green fluorescent protein (GFP)-tagged Dickeya sp. from stems or from leaves to underground parts of potato plants was studied in greenhouse experiments. Thirty days after stem inoculation, 90% of plants expressed symptoms at the stem base and 95% of plants showed browning of internal stem tissue. The GFP-tagged Dickeya sp. was detected by dilution plating in extracts of the stem interiors (100%), stem bases (90%), roots (80%), stolons (55%), and progeny tubers (24%). In roots, the GFP-tagged Dickeya sp. was found inside and between parenchyma cells whereas, in stems and stolons, the GFP-tagged Dickeya sp. was found in the xylem vessels and protoxylem cells. In progeny tubers, this strain was detected in the stolon end. Thirty days after leaf inoculation, the GFP-tagged Dickeya sp. was detected in extracts of 75% of the leaves, 88% of the petioles, 63% of the axils, and inside 25% of the stems taken 15 cm above the ground level. UV microscopy confirmed the presence of the GFP-tagged Dickeya sp. inside petioles and in the main leaf veins. No blackleg or aerial stem rot and no translocation of the GFP-tagged Dickeya sp. to underground plant parts was observed. The implications for contamination of progeny tubers are discussed.