Reaction of saprophytic bacteria from potato peel extracts and plant pathogenic bacteria in ELISA with antisera to Erwinia chrysanthemi (serogroup O1Ha)

The specificity of two antisera raised to whole cells ofErwinia chrysanthemi (Ech), serogroup O₁H_a, was studied in double antibody sandwich (DAS-) ELISA with 100 strains of different plant pathogenic bacteria (PPB), including 39 Ech strains, and of one of these antisera with 900 saprophytic bacteria isolated from extracts of potato peelings of Dutch seed potatoes grown in several production areas.All tested European Ech strains from potato reacted positively while no reactions were observed with any of the other plant pathogenic bacterial species. Two saprophytes (A254 and A256), both identified as pectinolyticPseudomonas fluorescens species, cross-reacted strongly with polyclonal antibodies against Ech. Non-specific reactions were found in DAS-ELISA with 16 saprophytes. The detection limits for the individual saprophytes varied between c. 10⁵ and 10⁹ cells.ml^–1. The non-specific reactions were also found with monoclonal antibodies (mca 2A4) against a proteinase K resistent epitope of Ech and with antisera against other plant pathogens including an antiserum against potato virus Y^N. The non-specific reactions were observed in DAS-ELISA, but not in Ouchterlony double diffusion or immunofluorescence colonystaining, whereas A254 and A256 reacted in all tests, but only with antibodies against Ech. When in making dilution series potato peel extracts were used instead of phosphate buffered saline with 0.1% Tween 20, the 14 non-specifically reacting saprophytes only reacted at concentrations of 10⁹ cells.ml^–1 or higher. Only one of these 14 saprophytes was able to multiply on injured potato tuber tissue.In contrast to most saprophytic strains, the saprophytes A254 and A256 reacted strongly in ELISA in dilutions series made with potato peel extracts. A256 was able to grow on potato tuber tissue but only under low oxygen conditions; A254 did not grow at all on potato tissue.Defatted milk powder or bovine serum albumin added to the dilution buffer for the enzymeconjugated antibodies, drastically reduced the non-specific reactions, but not the reactions with A254 and A256.To reduce the cross-reaction with A254, an Ech antiserum was absorbed with A254. This resulted in a substantial drop in antibody reaction with the homologous antigen in Ouchterlony double diffusion.     

Characterisation of Pseudomonas syringae Strains Associated with a Leaf Disease of Leek in Australia

A necrotic leaf disease of leek (Allium ampeloprasum Porrum Group) is reported in Australia for the first time. The fluorescent pseudomonad consistently associated with diseased tissue was identified as Pseudomonas syringae by LOPAT tests (+,−,−,−,+), carbon utilisation, bean and lemon inoculations and fatty acid methyl ester analysis. It was confirmed as P. syringae pv. porri by pathogenicity to leeks, bulb onions, spring onions, shallots and garlic, and by genetic analysis using 16S rDNA PCR, REP, ERIC and BOX PCR, and IS50 PCR. Comparison with reference strains of pv. porri from other countries showed similarity to known strains of pv. porri. The Australian leek strains were generally uniform in their biochemical reactions although three strains tested varied in their pathogenicity to other Allium spp. and varied from published data. All Australian strains shared the same genetic profile with strains from New Zealand, France and California. However, Japanese strains from leek and onion were distinct from the Australian strains and those from New Zealand, France and California. Data strongly support the hypothesis that the pathogen is seed-borne.     

Biochemical and molecular characterization of Bacillus amyloliquefaciens, B. subtilis and B. pumilus isolates with distinct antagonistic potential against Xanthomonas campestris pv. campestris

Fifty-one Bacillus isolates were characterized by fatty acid methyl ester (FAME) analysis; universal primer polymerase chain reaction (UP-PCR) fingerprinting; production of secondary metabolites and antagonistic activity against Xanthomonas campestris pv. campestris (causal agent of black rot in cabbage) in vitro and in vivo . Based on FAME analysis and/or PCR fingerprinting, the isolates were clustered into three different groups, named as Bacillus amyloliquefaciens , B . subtilis and B . pumilus . Seed treatment with Bacillus spp. generally reduced germination of seeds and incidence of black rot, but no relationship was found between the results of in vitro and in vivo experiments. The B .  amyloliquefaciens group contained isolates that were generally the most effective at reducing attack of black rot in vivo . The metabolic profiles of these isolates suggested that they produced surfactin, iturin, bacillomycine and/or azalomycin F. Isolates belonging to the B . subtilis group were mostly able to synthesize surfactin and arthrobactin. Surfactin, amphomycin, arthrobactin and valinomycin were generally found in culture extracts of isolates belonging to the B . pumilus group. No effect on growth of the pathogen was detected when the activity of filtered culture extracts and selected metabolites produced by the three different Bacillus species was tested in vitro against X . c . pv. campestris . However, inhibition was seen when bacterial liquid cultures were used. When the ability to colonize cabbage endophytically was examined for seven selected isolates with different antagonistic potential against black rot, it was found that the ability was related to the species and not to the antagonistic activity of the isolates.