New approach in detecting phytopathogenic bacteria by combined immunoisolation and immunoidentification assays¹

Abstract

Antibodies coated onto a suitable solid phase, e.g. polystyrene beads, rods for inoculating agar plates, or petri dishes, enable selective trapping of homologous bacteria and of immunologically related bacteria by immunoaffinity. After washing away unbound organisms, the bound organisms are desorbed and plated on a suitable medium. Selective immunoisolation has been demonstrated for various phytopathogenic bacteria including Corynebacterium michiganense ssp. michiganense, Erwinia chrysanthemi, E. carotovora ssp. atroseptica, Xanthomonas campestris pv. begoniae and Pseudomonas syringae pv. phaseolicola. Three serological methods are described for additional rapid immunoidentification of colonies directly on agar plates and for screening for cross-reacting microorganisms: (a) direct immunodiffusion on dilution plates, (b) agar mixed-antibody assay, (c) fluorescent antibody colony staining. Schemes are presented for increasing the reliability and sensitivity of sample screening for quality indexing of plant material, and for efficient screening and isolation of possible cross-reacting microorganisms to enable production of more specific antisera.

Components of partial resistance to potato blackleg caused by pectolytic Erwinia carotovora subsp. atroseptica and E. chrysanthemi

“…3. Decimal dilutions of 100 L aliquots of these suspensions were analysed for presence of Erwinia using the immunofluorescence colonystaining technique described by van Vuurde (1987). In short, in wells of 24-well microtiter plates (Greiner), dilutions of the samples were mixed with agar medium containing the appropriate antibiotic, and incubated for 48 h at 248C.…”

Potato blackleg: Epidemiology, host-pathogen interaction and control

“…None of the three methods available at present satisfies criteria 3 and 4; the viable count method based on the differential effect of temperature on growth and pectic enzyme production of the different erwinias is slow and it is not always easy to distinguish Eca in the presence of large numbers of either of the two other erwinias (P6rombelon et al, 1987); ELISA-based methods are not sensitive enough to detect populations <103 cells ml -~ tuber peel extract and fails to distinguish live from dead cells (Hyman and P6rombelon, 1990), and the fluorescent antibody colony staining method (Van Vuurde, 1987), although the best of the three methods, is slow. Difficulties are experienced in producing polyclonal antisera specific for all Eca serogroups or all biotypes of Echr.…”

Antibodies in plant science

“…Commonly aliquots of plant extracts are incubated on/in an enrichment medium to increase the numbers of pathogenic bacteria. Van Vuurde et al (1987) have developed an immunosorbent dilution-plating technique (lSDP) in which bacterial pathogens are trapped onto antibody-coated Petri dishes or inoculation rods. Unbound bacteria are removed by washing and those that remain are detected by incubating the trapped bacteria with selective media and allowing colonies to form.…”