Factors affecting survival of Clavibacter michiganesis subsp. sepedonicus in water

The survival of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, was studied in water, to assess the risks for dissemination of Cms via surface water and infection of potato crops by irrigation. Cms was able to survive for a maximum period of 7 days in non-sterile surface water at 10°C, a period during which Cms can be transported over long distances, but will also be strongly diluted. It is concluded that contamination of surface water with Cms can pose a threat on potato production only if aquatic host plants can multiply Cms in high densities. Survival of a fluidal and non-mucoid strain was also studied in sterile ditch water and simulated 'drainage water', in sterile MilliQ water, in tap water, in physiological salt and in artificial xylem fluid. In addition, the influence of temperature and low oxygen conditions on persistence of Cms in some of these diluents was studied. A maximum survival period of 35 days was found for Cms in sterile tap water at 20°C, independent of the strain used. In the other diluents survival periods ranged between 0 and 21 days. Relatively poor survival was found in MilliQ water and artificial xylem fluid. Low temperatures of 4°C do not favour survival as it does in soil. Oxygen depletion affected survival detrimentally. Survival periods determined by agar dilution plating and a direct viable counting method, based on the use of indicators for esterase activity and membrane integrity were similar. Therefore, it was concluded that under the experimental conditions studied, Cms did not form cells in a viable but non-culturable stat

Levensvatbaarheid van Ralstonia en Clavibacter meetbaar met behulp van RNA-detectiemethoden

Studie naar de epidemiologie van de bacterieziekten bruinrot (Ralstonia solacearum) en ringrot (Clavibacter michiganensis subsp. sepedonicus). De nieuwe methode is gebaseerd op amplificatie van 16S rRNA sequenties met behulp van NASBA (nucleic acid sequence based amplification). NASBA is een methode waarmee nucleïnezuren, maar in het bijzonder RNA-sequenties, efficiënt geamplificeerd kunnen worde

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. Methods and Results: Five methods were evaluated and fine-tuned for extraction of RNA from Cms. The most efficient non-commercial RNA extraction method included an enzymatic breakdown of the cell wall followed by a phenol extraction. AmpliDet RNA enabled detection of 10 000 molecules of purified rRNA per reaction and 100 cfu of Cms per reaction in more complex samples. Two primer pairs were tested with DNA and RNA purified from Cms. One primer pair was able to distinguish live from dead cells. Conclusions: An AmpliDet RNA was developed which enabled fast and specific detection of viable cells of Cms in complex substrates at a detection limit of 100 cfu per reactio

Stable recombinant alpaca antibodies for detection of Tulip virus X

For detection of the plant pathogenic Tulip virus X (TuVX), a panel of six recombinant antibodies was identified. To this end, a repertoire of variable domains from heavy-chain immunoglobulins (VHH) was cloned from an alpaca, which had been immunized with TuVX. Binding domains were selected by phage display and panning on immobilized TuVX particles. Recombinant VHH antibodies were tested for sensitivity in a sandwich ELISA, and were demonstrated to be readily able to distinguish TuVX-infected tulip leaf material from uninfected leafs. No cross-reactivity of the VHH antibodies to related flexiviridae was observed. Recombinant VHHs maintained their reactivity upon storage at ¿20°C for over a year. The effect of incubation at higher temperatures for prolonged time was studied. Two out of three VHH proteins retained activity after several weeks of storage at 37°

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